CRISPR gene editing#Recent events

In the early 2000s, German researchers began developing zinc finger nucleases (ZFNs), synthetic proteins whose DNA-binding domains enable them to create double-stranded breaks in DNA at specific points. ZFNs have a higher precision and the advantage of being smaller than Cas9, but ZFNs are not as commonly used as CRISPR-based methods. In 2010, synthetic nucleases called transcription activator-like effector nucleases (TALENs) provided an easier way to target a double-stranded break to a specific location on the DNA strand. Both zinc finger nucleases and TALENs require the design and creation of a custom protein for each targeted DNA sequence, which is a much more difficult and time-consuming process than that of designing guide RNAs. CRISPRs are much easier to design because the process requires synthesizing only a short RNA sequence, a procedure that is already widely used for many other molecular biology techniques (e.g. creating oligonucleotide primers).{{cite journal | vauthors = Young S | date=11 February 2014 |title= CRISPR and Other Genome Editing Tools Boost Medical Research and Gene Therapy's Reach |url=http://www.technologyreview.com/review/524451/genome-surgery|access-date=2014-04-13 |journal=MIT Technology Review}}

Whereas methods such as RNA interference (RNAi) do not fully suppress gene function, CRISPR, ZFNs, and TALENs provide full, irreversible gene knockout.{{cite journal | vauthors = Heidenreich M, Zhang F | title = Applications of CRISPR-Cas systems in neuroscience | journal = Nature Reviews. Neuroscience | volume = 17 | issue = 1 | pages = 36–44 | date = January 2016 | pmid = 26656253 | pmc = 4899966 | doi = 10.1038/nrn.2015.2 }} CRISPR can also target several DNA sites simultaneously simply by introducing different gRNAs. In addition, the costs of employing CRISPR are relatively low.{{cite journal | vauthors = Barrangou R, Doudna JA | title = Applications of CRISPR technologies in research and beyond | journal = Nature Biotechnology | volume = 34 | issue = 9 | pages = 933–941 | date = September 2016 | pmid = 27606440 | doi = 10.1038/nbt.3659 | s2cid = 21543486 }}{{cite journal | vauthors = Cox DB, Platt RJ, Zhang F | title = Therapeutic genome editing: prospects and challenges | journal = Nature Medicine | volume = 21 | issue = 2 | pages = 121–131 | date = February 2015 | pmid = 25654603 | pmc = 4492683 | doi = 10.1038/nm.3793 }}

In 2005, Alexander Bolotin at the French National Institute for Agricultural Research (INRA) discovered a CRISPR locus that contained novel Cas genes, significantly one that encoded a large protein known as Cas9.{{Cite web |date=2015-09-25 |title=CRISPR Timeline |url=https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/crispr-timeline |access-date=2023-12-08 |website=Broad Institute |language=en}}

In 2006, Eugene Koonin at the US National Center for Biotechnology information, NIH, proposed an explanation as to how CRISPR cascades as a bacterial immune system.

In 2007, Philippe Horvath at Danisco France SAS displayed experimentally how CRISPR systems are an adaptive immune system, and integrate new phage DNA into the CRISPR array, which is how they fight off the next wave of attacking phage.

In 2012, the research team led by professor Jennifer Doudna (University of California, Berkeley) and professor Emmanuelle Charpentier (Umeå University) were the first people to identify, disclose, and file a patent application for the CRISPR-Cas9 system needed to edit DNA. They also published their finding that CRISPR-Cas9 could be programmed with RNA to edit genomic DNA, now considered one of the most significant discoveries in the history of biology.

{{as of|2013|November}}, SAGE Labs (part of Horizon Discovery group) had exclusive rights from one of those companies to produce and sell genetically engineered rats and non-exclusive rights for mouse and rabbit models.{{cite web | url = http://www.genengnews.com/insight-and-intelligence/crispr-madness/77899947/ | title = CRISPR Madness | website = GEN | date = 2013-11-08 }} {{As of|2015|alt=By 2015}}, Thermo Fisher Scientific had licensed intellectual property from ToolGen to develop CRISPR reagent kits.{{Cite journal|last=Staff|date=1 April 2015|title=News: Products & Services|journal=Genetic Engineering & Biotechnology News|type=Paper|volume=35|issue=7|page=8|doi=10.1089/gen.35.21.05}}

{{as of|2014|December}}, patent rights to CRISPR were contested. Several companies formed to develop related drugs and research tools.{{cite web | url = https://www.technologyreview.com/2014/12/04/170211/who-owns-the-biggest-biotech-discovery-of-the-century/ | title = Who Owns the Biggest Biotech Discovery of the Century? There's a bitter fight over the patents for CRISPR, a breakthrough new form of DNA editing | website = MIT Technology Review | access-date = 25 February 2015 }} As companies ramped up financing, doubts as to whether CRISPR could be quickly monetized were raised.{{cite web | vauthors = Fye S | title=Genetic Rough Draft: Editas and CRISPR | url = http://atlasbusinessjournal.org/genetics/ | website = The Atlas Business Journal | access-date = 19 January 2016 }} In 2014, Feng Zhang of the Broad Institute of MIT and Harvard and nine others were awarded US patent number 8,697,359{{Cite web |title=CRISPR-Cas systems and methods for altering expression of gene products |url=https://patents.google.com/patent/US8697359B1/en |website=Google Patents}} over the use of CRISPR–Cas9 gene editing in eukaryotes. Although Charpentier and Doudna (referred to as CVC) were credited for the conception of CRISPR, the Broad Institute was the first to achieve a "reduction to practice" according to patent judges Sally Gardner Lane, James T. Moore and Deborah Katz.{{cite journal | vauthors = Shaffer C | title = Broad defeats Berkeley CRISPR patent | journal = Nature Biotechnology | volume = 40 | issue = 4 | pages = 445 | date = April 2022 | pmid = 35288688 | doi = 10.1038/d41587-022-00004-2 | s2cid = 247453528 }}

The first set of patents was awarded to the Broad team in 2015, prompting attorneys for the CVC group to request the first interference proceeding.{{cite journal | vauthors = | title = CRISPR patents to go on trial | journal = Nature Biotechnology | volume = 34 | issue = 2 | pages = 121 | date = February 2016 | pmid = 26849500 | doi = 10.1038/nbt0216-121a | s2cid = 205265912 | doi-access = free }} In February 2017, the US Patent Office ruled on a patent interference case brought by University of California with respect to patents issued to the Broad Institute, and found that the Broad patents, with claims covering the application of CRISPR-Cas9 in eukaryotic cells, were distinct from the inventions claimed by University of California.{{cite news | vauthors = Pollack A | title = Harvard and M.I.T. Scientists Win Gene-Editing Patent Fight | url = https://www.nytimes.com/2017/02/15/science/broad-institute-harvard-mit-gene-editing-patent.html | work = The New York Times | date = 15 February 2017 }}{{cite news | vauthors = Akst J |title=Broad Wins CRISPR Patent Interference Case | url = http://www.the-scientist.com/?articles.view/articleNo/48490/title/Broad-Wins-CRISPR-Patent-Interference-Case | work = The Scientist Magazine | date=February 15, 2017 }}{{cite news | vauthors = Noonan KE | title = PTAB Decides CRISPR Interference in Favor of Broad Institute – Their Reasoning | url = http://www.patentdocs.org/2017/02/ptab-decides-crispr-interference-in-favor-of-broad-institute-their-reasoning.html | work = Patent Docs | date = February 16, 2017 }}

Shortly after, University of California filed an appeal of this ruling.{{cite news | vauthors = Potenza A | title = UC Berkeley challenges decision that CRISPR patents belong to Broad Institute | url = https://www.theverge.com/2017/4/13/15278478/crispr-gene-editing-tool-patent-dispute-appeal-ucb-mit-broad | access-date=22 September 2017 | work=The Verge|date=April 13, 2017}}{{cite news| vauthors = Buhr S |title=The CRISPR patent battle is back on as UC Berkeley files an appeal|url=https://techcrunch.com/2017/07/26/the-crispr-patent-battle-is-back-on-as-uc-berkeley-files-an-appeal/|access-date=22 September 2017|work=TechCrunch|date=July 26, 2017}} In 2019 the second interference dispute was opened. This was in response to patent applications made by CVC that required the appeals board to determine the original inventor of the technology. The USPTO ruled in March 2022 against UC, stating that the Broad Institute were first to file. The decision affected many of the licensing agreements for the CRISPR editing technology that was licensed from UC Berkeley. UC stated its intent to appeal the USPTO's ruling.{{cite web | url = https://www.theverge.com/2022/3/1/22956326/crispr-patent-uc-berkeley-broad-institute-nobel | title = UC Berkeley loses CRISPR patent case | vauthors = Westman N | date = March 1, 2022 | access-date = March 6, 2022 | work = The Verge }}

In March 2017, the European Patent Office (EPO) announced its intention to allow claims for editing all types of cells to Max-Planck Institute in Berlin, University of California, and University of Vienna,{{cite news| vauthors = Akst J |title=UC Berkeley Receives CRISPR Patent in Europe|url=http://www.the-scientist.com/?articles.view/articleNo/48987/title/UC-Berkeley-Receives-CRISPR-Patent-in-Europe/|access-date=22 September 2017|work=The Scientist|date=March 24, 2017}} and in August 2017, the EPO announced its intention to allow CRISPR claims in a patent application that MilliporeSigma had filed.{{cite news| vauthors = Philippidis A |title=MilliporeSigma to Be Granted European Patent for CRISPR Technology|work=Genetic Engineering & Biotechology News|date=August 7, 2017|url=http://www.genengnews.com/gen-news-highlights/milliporesigma-to-be-granted-european-patent-for-crispr-technology/81254776|access-date=22 September 2017}} {{as of|2017|August}} the patent situation in Europe was complex, with MilliporeSigma, ToolGen, Vilnius University, and Harvard contending for claims, along with University of California and Broad.{{cite journal | vauthors = Cohen J | title = CRISPR patent battle in Europe takes a 'wild' twist with surprising player|journal=Science|date=4 August 2017 | doi = 10.1126/science.aan7211 | url = https://www.science.org/content/article/crispr-patent-battle-europe-takes-wild-twist-surprising-player | url-access = subscription }}

In July 2018, the ECJ ruled that gene editing for plants was a sub-category of GMO foods and therefore that the CRISPR technique would henceforth be regulated in the European Union by their rules and regulations for GMOs.{{cite news |title=Top EU court: GMO rules cover plant gene editing technique |url=https://www.reuters.com/article/us-eu-court-gmo/top-eu-court-gmo-rules-cover-plant-gene-editing-technique-idUSKBN1KF15L |publisher=Retuers |date=25 July 2018}}

In February 2020, a US trial showed safe CRISPR gene editing on three cancer patients.{{Cite web|url=https://www.sciencealert.com/researchers-genetically-alter-the-immune-system-of-cancer-patients-without-side-effect|title=US Trial Shows 3 Cancer Patients Had Their Genomes Altered Safely by CRISPR|last=AFP|website=ScienceAlert|date=7 February 2020 |language=en-gb|access-date=2020-02-09}}

In October 2020, researchers Emmanuelle Charpentier and Jennifer Doudna were awarded the Nobel Prize in Chemistry for their work in this field.{{Cite web|url=https://www.forbes.com/sites/jvchamary/2020/10/07/crispr-genome-editing-nobel-prize/#18cbb9462d32|title=These Scientists Deserved A Nobel Prize, But Didn't Discover Crispr| vauthors = Chamary JV |website=Forbes|language=en-gb|access-date=2020-07-10}}{{Cite web| vauthors = Fischman J |title=Nobel Prize in Chemistry Goes to Discovery of 'Genetic Scissors' Called CRISPR/Cas9|url=https://www.scientificamerican.com/article/nobel-prize-in-chemistry-goes-to-discovery-of-genetic-scissors-called-crispr-cas911/|access-date=2021-03-24|website=Scientific American|language=en}} They made history as the first two women to share this award without a male contributor.{{Cite news|date=2020-10-07|title=Two women share chemistry Nobel in historic win for 'genetic scissors'|language=en-GB|work=BBC News|url=https://www.bbc.com/news/science-environment-54432589|access-date=2020-12-06}}{{cite web | vauthors = Owens R | title=Nobel prize: who gets left out? | website=The Conversation | date=8 October 2020 | url=http://theconversation.com/nobel-prize-who-gets-left-out-147759 | access-date=13 December 2021}}

In June 2021, the first, small clinical trial of intravenous CRISPR gene editing in humans concluded with promising results.{{cite news | vauthors = Kaiser J |title=CRISPR injected into the blood treats a genetic disease for first time |url=https://www.science.org/content/article/crispr-injected-blood-treats-genetic-disease-first-time |access-date=11 July 2021 |work=Science {{!}} AAAS |date=26 June 2021 |language=en}}{{cite journal | vauthors = Gillmore JD, Gane E, Taubel J, Kao J, Fontana M, Maitland ML, Seitzer J, O'Connell D, Walsh KR, Wood K, Phillips J, Xu Y, Amaral A, Boyd AP, Cehelsky JE, McKee MD, Schiermeier A, Harari O, Murphy A, Kyratsous CA, Zambrowicz B, Soltys R, Gutstein DE, Leonard J, Sepp-Lorenzino L, Lebwohl D | title = CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis | journal = The New England Journal of Medicine | volume = 385 | issue = 6 | pages = 493–502 | date = August 2021 | pmid = 34215024 | doi = 10.1056/NEJMoa2107454 | s2cid = 235722446 | doi-access = free }}

In September 2021, the first CRISPR-edited food went on public sale in Japan. Tomatoes were genetically modified for around five times the normal amount of possibly calming{{cite journal | vauthors = Boonstra E, de Kleijn R, Colzato LS, Alkemade A, Forstmann BU, Nieuwenhuis S | title = Neurotransmitters as food supplements: the effects of GABA on brain and behavior | journal = Frontiers in Psychology | volume = 6 | pages = 1520 | date = 6 October 2015 | pmid = 26500584 | pmc = 4594160 | doi = 10.3389/fpsyg.2015.01520 | doi-access = free }} GABA.{{cite news |title=Tomato In Japan Is First CRISPR-Edited Food In The World To Go On Sale |url=https://www.iflscience.com/plants-and-animals/tomato-in-japan-is-first-crispredited-food-to-go-on-sale-/ |access-date=18 October 2021 |work=IFLScience |language=en}} CRISPR was first applied in tomatoes in 2014.{{cite journal | vauthors = Wang T, Zhang H, Zhu H | title = CRISPR technology is revolutionizing the improvement of tomato and other fruit crops | journal = Horticulture Research | volume = 6 | issue = 1 | pages = 77 | date = 15 June 2019 | pmid = 31240102 | pmc = 6570646 | doi = 10.1038/s41438-019-0159-x | bibcode = 2019HorR....6...77W }}

In December 2021, it was reported that the first CRISPR-gene-edited marine animal/seafood and second set of CRISPR-edited food has gone on public sale in Japan: two fish of which one species grows to twice the size of natural specimens due to disruption of leptin, which controls appetite, and the other grows to 1.2 times the natural average size with the same amount of food due to disabled myostatin, which inhibits muscle growth.{{cite journal | vauthors = | title = Japan embraces CRISPR-edited fish | journal = Nature Biotechnology | volume = 40 | issue = 1 | pages = 10 | date = January 2022 | pmid = 34969964 | doi = 10.1038/s41587-021-01197-8 | s2cid = 245593283 }}{{cite news |title=Startup hopes genome-edited pufferfish will be a hit in 2022 |url=https://www.japantimes.co.jp/life/2022/01/05/food/startup-hopes-genome-edited-pufferfish-will-hit-2022/ |access-date=17 January 2022 |work=The Japan Times |date=5 January 2022 |archive-date=17 January 2022 |archive-url=https://web.archive.org/web/20220117022646/https://www.japantimes.co.jp/life/2022/01/05/food/startup-hopes-genome-edited-pufferfish-will-hit-2022/ |url-status=dead }}{{cite news |title=Gene-edited sea bream set for sale in Japan |url=https://thefishsite.com/articles/gene-edited-sea-bream-set-for-sale-in-japan |work=thefishsite.com |language=en}}

A 2022 study has found that knowing more about CRISPR tomatoes had a strong effect on the participants' preference. "Almost half of the 32 participants from Germany who are scientists demonstrated constant choices, while the majority showed increased willingness to buy CRISPR tomatoes, mostly non-scientists."{{Cite journal | vauthors = Götz L, Svanidze M, Tissier A, Brand A |date= January 2022 |title=Consumers' Willingness to Buy CRISPR Gene-Edited Tomatoes: Evidence from a Choice Experiment Case Study in Germany | journal = Sustainability |volume=14 |issue=2 |pages=971 |doi=10.3390/su14020971 |doi-access= free |bibcode= 2022Sust...14..971G |hdl=10419/249208 |hdl-access=free }}{{Cite web|title=Are Consumers Willing to Buy CRISPR Tomatoes?|url= http://www.isaaa.org/kc/cropbiotechupdate/article/default.asp?ID=19225|access-date=2022-02-21 |website=Crop Biotech Update|language=en}}

In May 2021, UC Berkeley announced their intent to auction non-fungible tokens of both the patent for CRISPR gene editing as well as cancer immunotherapy. However, the university would in this case retain ownership of the patents.{{cite web |url=https://news.berkeley.edu/2021/05/27/uc-berkeley-will-auction-nfts-of-nobel-prize-winning-inventions-to-fund-research/ |title=UC Berkeley Will Auction NFTs for 2 Nobel Prize Patents | vauthors = Whitford E |date=2021-05-28 |publisher=Inside Higher Ed |access-date=2023-02-21}}{{cite book | vauthors = Sestino A, Guido G, Peluso AM |date=2022 |title=Non-Fungible Tokens (NFTs). Examining the Impact on Consumers and Marketing Strategies |url= |location= |publisher= |doi=10.1007/978-3-031-07203-1 |page=28|isbn=978-3-031-07202-4 |s2cid=250238540 }} 85 % of funds gathered through the sale of the collection named The Fourth Pillar were to be used to finance research.{{cite web |url=https://www.nytimes.com/2021/05/27/science/nobel-prize-nft-berkeley.html |title=You Can Buy a Piece of a Nobel Prize-Winning Discovery | vauthors = Chang K |date=2021-05-27 |work=New York Times |access-date=2023-02-21}}{{cite journal | vauthors = Trautman LJ |date=2022 |title=Virtual Art and Non-Fungible Tokens |journal=Hofstra Law Review |volume=50 |issue=361 |pages=369 f |doi=10.2139/ssrn.3814087|s2cid=234830426 |url=https://scholarlycommons.law.hofstra.edu/context/hlr/article/3172/viewcontent/5____virtual_art____ARTICLE____fourth_article__pages_361_to_426.pdf }} It sold in June 2022 for 22 Ether, which was around {{Currency|54000|US}} at the time.{{cite journal | vauthors = Jones N |date=2021-06-18 |title=How scientists are embracing NFTs |journal=Nature |volume=594 |issue=7864 |pages=482 |doi=10.1038/d41586-021-01642-3|pmid=34145410 |bibcode=2021Natur.594..481J |s2cid=235481285 |doi-access=free }}

In November 2023, the United Kingdom's Medicines and Healthcare products Regulatory Agency (MHRA) became the first in the world to approve the use of the first drug based on CRISPR gene editing, Casgevy, to treat sickle-cell anemia and beta thalassemia. Casgevy, or exagamglogene autotemcel, directly acts on the genes of the stem cells inside the patient's bones, having them produce healthy red blood cells. This treatment thus avoids the need for regular, costly blood transfusions.

In December 2023, the FDA approved the first gene therapy in the US to treat patients with Sickle Cell Disease (SCD). The FDA approved two milestone treatments, Casgevy and Lyfgenia, representing the first cell-based gene therapies for the treatment of SCD.{{Cite web | author = Office of the Commissioner |date=2023-12-08 |title=FDA Approves First Gene Therapies to Treat Patients with Sickle Cell Disease |url=https://www.fda.gov/news-events/press-announcements/fda-approves-first-gene-therapies-treat-patients-sickle-cell-disease |access-date=2023-12-14 |website=FDA |language=en}}

File:DNA Repair.png

CRISPR-Cas9 genome editing uses a Type II CRISPR system. This system includes a ribonucleoprotein (RNP), consisting of Cas9, crRNA, and tracrRNA, along with an optional DNA repair template.

File:CRISPR overview - en.svg

CRISPR-Cas9 often employs plasmids that code for the RNP components to transfect the target cells, or the RNP is assembled before addition to the cells via nucleofection. The main components of this plasmid are displayed in the image and listed in the table. The crRNA is uniquely designed for each application, as this is the sequence that Cas9 uses to identify and directly bind to specific sequences within the host cell's DNA. The crRNA must bind only where editing is desired. The repair template is also uniquely designed for each application, as it must complement to some degree the DNA sequences on either side of the cut and also contain whatever sequence is desired for insertion into the host genome.

Multiple crRNAs and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA).{{cite thesis |url=https://archive.org/details/LyJosephP201311PhDThesis |title=Discovering Genes Responsible for Kidney Diseases | vauthors = Ly J |year=2013 |type=Ph.D. |publisher=University of Toronto |access-date=26 December 2016}} This sgRNA can be included alongside the gene that codes for the Cas9 protein and made into a plasmid in order to be transfected into cells. Many online tools are available to aid in designing effective sgRNA sequences.{{cite journal | vauthors = Mohr SE, Hu Y, Ewen-Campen B, Housden BE, Viswanatha R, Perrimon N | title = CRISPR guide RNA design for research applications | journal = The FEBS Journal | volume = 283 | issue = 17 | pages = 3232–3238 | date = September 2016 | pmid = 27276584 | pmc = 5014588 | doi = 10.1111/febs.13777 }}{{cite journal | vauthors = Brazelton VA, Zarecor S, Wright DA, Wang Y, Liu J, Chen K, Yang B, Lawrence-Dill CJ | title = A quick guide to CRISPR sgRNA design tools | journal = GM Crops & Food | volume = 6 | issue = 4 | pages = 266–276 | date = 2015 | pmid = 26745836 | pmc = 5033207 | doi = 10.1080/21645698.2015.1137690 }}

File:CRISPR transfection.png

{{Further|CRISPR#Cas genes and CRISPR subtypes}}

File:PAMs of different CRISPR nucleases.svg

Alternative proteins to Cas9 include the following:

CRISPR-Cas9 offers a high degree of fidelity and relatively simple construction. It depends on two factors for its specificity: the target sequence and the protospacer adjacent motif (PAM) sequence. The target sequence is 20 bases long as part of each CRISPR locus in the crRNA array.{{cite journal | vauthors = Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F | title = Genome engineering using the CRISPR-Cas9 system | journal = Nature Protocols | volume = 8 | issue = 11 | pages = 2281–2308 | date = November 2013 | pmid = 24157548 | pmc = 3969860 | doi = 10.1038/nprot.2013.143 | hdl = 1721.1/102943 }} A typical crRNA array has multiple unique target sequences. Cas9 proteins select the correct location on the host's genome by utilizing the sequence to bond with base pairs on the host DNA. The sequence is not part of the Cas9 protein and as a result is customizable and can be independently synthesized.{{cite journal | vauthors = Horvath P, Barrangou R | title = CRISPR/Cas, the immune system of bacteria and archaea | journal = Science | volume = 327 | issue = 5962 | pages = 167–170 | date = January 2010 | pmid = 20056882 | doi = 10.1126/science.1179555 | s2cid = 17960960 | bibcode = 2010Sci...327..167H }}{{cite journal | vauthors = Bialk P, Rivera-Torres N, Strouse B, Kmiec EB | title = Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems | journal = PLOS ONE | volume = 10 | issue = 6 | pages = e0129308 | date = 2015-06-08 | pmid = 26053390 | pmc = 4459703 | doi = 10.1371/journal.pone.0129308 | doi-access = free | bibcode = 2015PLoSO..1029308B }}

The PAM sequence on the host genome is recognized by Cas9. Cas9 cannot be easily modified to recognize a different PAM sequence. However, this is ultimately not too limiting, as it is typically a very short and nonspecific sequence that occurs frequently at many places throughout the genome (e.g. the SpCas9 PAM sequence is 5'-NGG-3' and in the human genome occurs roughly every 8 to 12 base pairs).

Once these sequences have been assembled into a plasmid and transfected into cells, the Cas9 protein with the help of the crRNA finds the correct sequence in the host cell's DNA and – depending on the Cas9 variant – creates a single- or double-stranded break at the appropriate location in the DNA.{{cite journal | vauthors = Sander JD, Joung JK | title = CRISPR-Cas systems for editing, regulating and targeting genomes | journal = Nature Biotechnology | volume = 32 | issue = 4 | pages = 347–355 | date = April 2014 | pmid = 24584096 | pmc = 4022601 | doi = 10.1038/nbt.2842 }}

Properly spaced single-stranded breaks in the host DNA can trigger homology directed repair, which is less error-prone than the non-homologous end joining or theta-mediated end joining that typically follows a double-stranded break. Providing a DNA repair template allows for the insertion of a specific DNA sequence at an exact location within the genome. The repair template should extend 40 to 90 base pairs beyond the Cas9-induced DNA break. The goal is for the cell's native HDR process to utilize the provided repair template and thereby incorporate the new sequence into the genome. Once incorporated, this new sequence is now part of the cell's genetic material and passes into its daughter cells. Combined transient inhibition of NHEJ and TMEJ by a small molecule and siRNAs can increase HDR efficiency to up to 93% and simultaneously prevent off-target editing.{{cite journal | vauthors = Riesenberg S, Kanis P, Macak D, Wollny D, Düsterhöft D, Kowalewski J, Helmbrecht N, Maricic T, Pääbo S | title = Efficient high-precision homology-directed repair-dependent genome editing by HDRobust | journal = Nature Methods | volume = 20 | issue = 9 | pages = 1388–1399 | date = September 2023 | pmid = 37474806 | pmc = 10482697 | doi = 10.1038/s41592-023-01949-1 | doi-access = free }}

{{See also|Transfection}}

Delivery of Cas9, sgRNA, and associated complexes into cells can occur via viral and non-viral systems. Electroporation of DNA, RNA, or ribonucleocomplexes is a common technique, though it can result in harmful effects on the target cells.{{cite journal | vauthors = Lino CA, Harper JC, Carney JP, Timlin JA | title = Delivering CRISPR: a review of the challenges and approaches | journal = Drug Delivery | volume = 25 | issue = 1 | pages = 1234–1257 | date = November 2018 | pmid = 29801422 | pmc = 6058482 | doi = 10.1080/10717544.2018.1474964 }} Chemical transfection techniques utilizing lipids and peptides have also been used to introduce sgRNAs in complex with Cas9 into cells.{{cite journal | vauthors = Li L, Hu S, Chen X | title = Non-viral delivery systems for CRISPR/Cas9-based genome editing: Challenges and opportunities | journal = Biomaterials | volume = 171 | pages = 207–218 | date = July 2018 | pmid = 29704747 | pmc = 5944364 | doi = 10.1016/j.biomaterials.2018.04.031 }}{{cite journal | vauthors = Jain PK, Lo JH, Rananaware S, Downing M, Panda A, Tai M, Raghavan S, Fleming HE, Bhatia SN | title = Non-viral delivery of CRISPR/Cas9 complex using CRISPR-GPS nanocomplexes | journal = Nanoscale | volume = 11 | issue = 44 | pages = 21317–21323 | date = November 2019 | pmid = 31670340 | pmc = 7709491 | doi = 10.1039/C9NR01786K }} Nanoparticle-based delivery has also been used for transfection.{{cite journal | vauthors = Yip BH | title = Recent Advances in CRISPR/Cas9 Delivery Strategies | journal = Biomolecules | volume = 10 | issue = 6 | pages = 839 | date = May 2020 | pmid = 32486234 | pmc = 7356196 | doi = 10.3390/biom10060839 | doi-access = free }} Types of cells that are more difficult to transfect (e.g., stem cells, neurons, and hematopoietic cells) require more efficient delivery systems, such as those based on lentivirus (LVs), adenovirus (AdV), and adeno-associated virus (AAV).{{cite journal | vauthors = Bak RO, Porteus MH | title = CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors | journal = Cell Reports | volume = 20 | issue = 3 | pages = 750–756 | date = July 2017 | pmid = 28723575 | pmc = 5568673 | doi = 10.1016/j.celrep.2017.06.064 }}{{cite journal | vauthors = Schmidt F, Grimm D | title = CRISPR genome engineering and viral gene delivery: a case of mutual attraction | journal = Biotechnology Journal | volume = 10 | issue = 2 | pages = 258–272 | date = February 2015 | pmid = 25663455 | doi = 10.1002/biot.201400529 | s2cid = 37653318 }}{{cite web| title = CRISPR 101: Mammalian Expression Systems and Delivery Methods | vauthors = Waxmonsky N | url = https://blog.addgene.org/crispr-101-mammalian-expression-systems-and-delivery-methods | date = 24 September 2015 | access-date = 11 June 2018 }}

Efficiency of CRISPR-Cas9 has been found to greatly increase when various components of the system including the entire CRISPR/Cas9 structure to Cas9-gRNA complexes delivered in assembled form rather than using transgenics.{{cite journal | vauthors = Mishra T, Bhardwaj V, Ahuja N, Gadgil P, Ramdas P, Shukla S, Chande A | title = Improved loss-of-function CRISPR-Cas9 genome editing in human cells concomitant with inhibition of TGF-β signaling | journal = Molecular Therapy. Nucleic Acids | volume = 28 | pages = 202–218 | date = June 2022 | pmid = 35402072 | pmc = 8961078 | doi = 10.1016/j.omtn.2022.03.003 }}{{cite journal | vauthors = Adli M | title = The CRISPR tool kit for genome editing and beyond | journal = Nature Communications | volume = 9 | issue = 1 | pages = 1911 | date = May 2018 | pmid = 29765029 | pmc = 5953931 | doi = 10.1038/s41467-018-04252-2 | bibcode = 2018NatCo...9.1911A }} This has found particular value in genetically modified crops for mass commercialization.{{cite journal | vauthors = Svitashev S, Schwartz C, Lenderts B, Young JK, Mark Cigan A | title = Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes | journal = Nature Communications | volume = 7 | issue = 1 | pages = 13274 | date = November 2016 | pmid = 27848933 | pmc = 5116081 | doi = 10.1038/ncomms13274 | bibcode = 2016NatCo...713274S }}{{cite journal | vauthors = Liang Z, Chen K, Li T, Zhang Y, Wang Y, Zhao Q, Liu J, Zhang H, Liu C, Ran Y, Gao C | title = Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes | journal = Nature Communications | volume = 8 | issue = 1 | pages = 14261 | date = January 2017 | pmid = 28098143 | pmc = 5253684 | doi = 10.1038/ncomms14261 | s2cid = 17028472 | bibcode = 2017NatCo...814261L }} Since the host's replication machinery is not needed to produce these proteins, the chance of the recognizing sequence of the sgRNA is almost none, decreasing the chance of off-target effects.

Further improvements and variants of the CRISPR-Cas9 system have focused on introducing more control into its use. Specifically, the research aimed at improving this system includes improving its specificity, its efficiency, and the granularity of its editing power. Techniques can further be divided and classified by the component of the system they modify. These include using different variants or novel creations of the Cas protein, using an altogether different effector protein, modifying the sgRNA, or using an algorithmic approach to identify existing optimal solutions.

Specificity is an important aspect to improve the CRISPR-Cas9 system because the off-target effects it generates have serious consequences for the genome of the cell and invokes caution for its use. Minimizing off-target effects is thus maximizing the safety of the system. Novel variations of Cas9 proteins that increase specificity include effector proteins with comparable efficiency and specificity to the original SpCas9 that are able to target the previously untargetable sequences and a variant that has virtually no off-target mutations.{{cite journal | vauthors = Cui Z, Tian R, Huang Z, Jin Z, Li L, Liu J, Huang Z, Xie H, Liu D, Mo H, Zhou R, Lang B, Meng B, Weng H, Hu Z | title = FrCas9 is a CRISPR/Cas9 system with high editing efficiency and fidelity | journal = Nature Communications | volume = 13 | issue = 1 | pages = 1425 | date = March 2022 | pmid = 35301321 | pmc = 8931148 | doi = 10.1038/s41467-022-29089-8 | bibcode = 2022NatCo..13.1425C }}{{cite journal | vauthors = Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK | title = High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects | journal = Nature | volume = 529 | issue = 7587 | pages = 490–495 | date = January 2016 | pmid = 26735016 | pmc = 4851738 | doi = 10.1038/nature16526 | bibcode = 2016Natur.529..490K }} Research has also been conducted in engineering new Cas9 proteins, including some that partially replace RNA nucleotides in crRNA with DNA and a structure-guided Cas9 mutant generating procedure that all had reduced off-target effects.{{cite journal | vauthors = Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F | title = Rationally engineered Cas9 nucleases with improved specificity | journal = Science | volume = 351 | issue = 6268 | pages = 84–88 | date = January 2016 | pmid = 26628643 | pmc = 4714946 | doi = 10.1126/science.aad5227 | bibcode = 2016Sci...351...84S }}{{cite journal | vauthors = Yin H, Song CQ, Suresh S, Kwan SY, Wu Q, Walsh S, Ding J, Bogorad RL, Zhu LJ, Wolfe SA, Koteliansky V, Xue W, Langer R, Anderson DG | title = Partial DNA-guided Cas9 enables genome editing with reduced off-target activity | journal = Nature Chemical Biology | volume = 14 | issue = 3 | pages = 311–316 | date = March 2018 | pmid = 29377001 | pmc = 5902734 | doi = 10.1038/nchembio.2559 }} Iteratively truncated sgRNAs and highly stabilized gRNAs have been shown to also decrease off-target effects.{{cite journal | vauthors = Riesenberg S, Helmbrecht N, Kanis P, Maricic T, Pääbo S | title = Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage | journal = Nature Communications | volume = 13 | issue = 1 | pages = 489 | date = January 2022 | pmid = 35078986 | pmc = 8789806 | doi = 10.1038/s41467-022-28137-7 | s2cid = 246281892 | bibcode = 2022NatCo..13..489R }}{{cite journal | vauthors = Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK | title = Improving CRISPR-Cas nuclease specificity using truncated guide RNAs | journal = Nature Biotechnology | volume = 32 | issue = 3 | pages = 279–284 | date = March 2014 | pmid = 24463574 | pmc = 3988262 | doi = 10.1038/nbt.2808 }} Computational methods including machine learning have been used to predict the affinity of and create unique sequences for the system to maximize specificity for given targets.{{cite journal | vauthors = Thean DG, Chu HY, Fong JH, Chan BK, Zhou P, Kwok CC, Chan YM, Mak SY, Choi GC, Ho JW, Zheng Z, Wong AS | title = Machine learning-coupled combinatorial mutagenesis enables resource-efficient engineering of CRISPR-Cas9 genome editor activities | journal = Nature Communications | volume = 13 | issue = 1 | pages = 2219 | date = April 2022 | pmid = 35468907 | pmc = 9039034 | doi = 10.1038/s41467-022-29874-5 | bibcode = 2022NatCo..13.2219T }}{{cite journal | vauthors = Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, Kim JS | title = Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases | journal = Genome Research | volume = 24 | issue = 1 | pages = 132–141 | date = January 2014 | pmid = 24253446 | pmc = 3875854 | doi = 10.1101/gr.162339.113 }}

Several variants of CRISPR-Cas9 allow gene activation or genome editing with an external trigger such as light or small molecules.{{cite journal | vauthors = Oakes BL, Nadler DC, Flamholz A, Fellmann C, Staahl BT, Doudna JA, Savage DF | title = Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch | journal = Nature Biotechnology | volume = 34 | issue = 6 | pages = 646–651 | date = June 2016 | pmid = 27136077 | pmc = 4900928 | doi = 10.1038/nbt.3528 }}{{cite journal | vauthors = Nuñez JK, Harrington LB, Doudna JA | title = Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering | journal = ACS Chemical Biology | volume = 11 | issue = 3 | pages = 681–688 | date = March 2016 | pmid = 26857072 | doi = 10.1021/acschembio.5b01019 }}{{cite journal | vauthors = Zhou W, Deiters A | title = Conditional Control of CRISPR/Cas9 Function | journal = Angewandte Chemie | volume = 55 | issue = 18 | pages = 5394–5399 | date = April 2016 | pmid = 26996256 | doi = 10.1002/anie.201511441 | doi-access = free }} These include photoactivatable CRISPR systems developed by fusing light-responsive protein partners with an activator domain and a dCas9 for gene activation,{{cite journal | vauthors = Polstein LR, Gersbach CA | title = A light-inducible CRISPR-Cas9 system for control of endogenous gene activation | journal = Nature Chemical Biology | volume = 11 | issue = 3 | pages = 198–200 | date = March 2015 | pmid = 25664691 | pmc = 4412021 | doi = 10.1038/nchembio.1753 }}{{cite journal | vauthors = Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M | title = CRISPR-Cas9-based photoactivatable transcription system | journal = Chemistry & Biology | volume = 22 | issue = 2 | pages = 169–174 | date = February 2015 | pmid = 25619936 | doi = 10.1016/j.chembiol.2014.12.011 | doi-access = free }} or by fusing similar light-responsive domains with two constructs of split-Cas9,{{cite journal | vauthors = Wright AV, Sternberg SH, Taylor DW, Staahl BT, Bardales JA, Kornfeld JE, Doudna JA | title = Rational design of a split-Cas9 enzyme complex | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 112 | issue = 10 | pages = 2984–2989 | date = March 2015 | pmid = 25713377 | pmc = 4364227 | doi = 10.1073/pnas.1501698112 | doi-access = free | bibcode = 2015PNAS..112.2984W }}{{cite journal | vauthors = Nihongaki Y, Kawano F, Nakajima T, Sato M | title = Photoactivatable CRISPR-Cas9 for optogenetic genome editing | journal = Nature Biotechnology | volume = 33 | issue = 7 | pages = 755–760 | date = July 2015 | pmid = 26076431 | doi = 10.1038/nbt.3245 | s2cid = 205281536 }} or by incorporating caged unnatural amino acids into Cas9,{{cite journal | vauthors = Hemphill J, Borchardt EK, Brown K, Asokan A, Deiters A | title = Optical Control of CRISPR/Cas9 Gene Editing | journal = Journal of the American Chemical Society | volume = 137 | issue = 17 | pages = 5642–5645 | date = May 2015 | pmid = 25905628 | pmc = 4919123 | doi = 10.1021/ja512664v | bibcode = 2015JAChS.137.5642H }} or by modifying the guide RNAs with photocleavable complements for genome editing.{{cite journal | vauthors = Jain PK, Ramanan V, Schepers AG, Dalvie NS, Panda A, Fleming HE, Bhatia SN | title = Development of Light-Activated CRISPR Using Guide RNAs with Photocleavable Protectors | journal = Angewandte Chemie | volume = 55 | issue = 40 | pages = 12440–12444 | date = September 2016 | pmid = 27554600 | pmc = 5864249 | doi = 10.1002/anie.201606123 }}

Methods to control genome editing with small molecules include an allosteric Cas9, with no detectable background editing, that will activate binding and cleavage upon the addition of 4-hydroxytamoxifen (4-HT), 4-HT responsive intein-linked Cas9,{{cite journal | vauthors = Davis KM, Pattanayak V, Thompson DB, Zuris JA, Liu DR | title = Small molecule-triggered Cas9 protein with improved genome-editing specificity | journal = Nature Chemical Biology | volume = 11 | issue = 5 | pages = 316–318 | date = May 2015 | pmid = 25848930 | pmc = 4402137 | doi = 10.1038/nchembio.1793 }} or a Cas9 that is 4-HT responsive when fused to four ERT2 domains.{{cite journal | vauthors = Liu KI, Ramli MN, Woo CW, Wang Y, Zhao T, Zhang X, Yim GR, Chong BY, Gowher A, Chua MZ, Jung J, Lee JH, Tan MH | title = A chemical-inducible CRISPR-Cas9 system for rapid control of genome editing | journal = Nature Chemical Biology | volume = 12 | issue = 11 | pages = 980–987 | date = November 2016 | pmid = 27618190 | doi = 10.1038/nchembio.2179 | s2cid = 33891039 }} Intein-inducible split-Cas9 allows dimerization of Cas9 fragments{{cite journal | vauthors = Truong DJ, Kühner K, Kühn R, Werfel S, Engelhardt S, Wurst W, Ortiz O | title = Development of an intein-mediated split-Cas9 system for gene therapy | journal = Nucleic Acids Research | volume = 43 | issue = 13 | pages = 6450–6458 | date = July 2015 | pmid = 26082496 | pmc = 4513872 | doi = 10.1093/nar/gkv601 }} and rapamycin-inducible split-Cas9 system developed by fusing two constructs of split-Cas9 with FRB and FKBP fragments.{{cite journal | vauthors = Zetsche B, Volz SE, Zhang F | title = A split-Cas9 architecture for inducible genome editing and transcription modulation | journal = Nature Biotechnology | volume = 33 | issue = 2 | pages = 139–142 | date = February 2015 | pmid = 25643054 | pmc = 4503468 | doi = 10.1038/nbt.3149 }} Other studies have been able to induce transcription of Cas9 with a small molecule, doxycycline.{{cite journal | vauthors = González F, Zhu Z, Shi ZD, Lelli K, Verma N, Li QV, Huangfu D | title = An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells | journal = Cell Stem Cell | volume = 15 | issue = 2 | pages = 215–226 | date = August 2014 | pmid = 24931489 | pmc = 4127112 | doi = 10.1016/j.stem.2014.05.018 }}{{cite journal | vauthors = Dow LE, Fisher J, O'Rourke KP, Muley A, Kastenhuber ER, Livshits G, Tschaharganeh DF, Socci ND, Lowe SW | title = Inducible in vivo genome editing with CRISPR-Cas9 | journal = Nature Biotechnology | volume = 33 | issue = 4 | pages = 390–394 | date = April 2015 | pmid = 25690852 | pmc = 4390466 | doi = 10.1038/nbt.3155 }} Small molecules can also be used to improve homology directed repair,{{cite journal | vauthors = Yu C, Liu Y, Ma T, Liu K, Xu S, Zhang Y, Liu H, La Russa M, Xie M, Ding S, Qi LS | title = Small molecules enhance CRISPR genome editing in pluripotent stem cells | journal = Cell Stem Cell | volume = 16 | issue = 2 | pages = 142–147 | date = February 2015 | pmid = 25658371 | pmc = 4461869 | doi = 10.1016/j.stem.2015.01.003 }} often by inhibiting the non-homologous end joining pathway and/or the theta-mediated end-joining pathway.{{cite journal | vauthors = Schimmel J, Muñoz-Subirana N, Kool H, van Schendel R, van der Vlies S, Kamp JA, de Vrij FM, Kushner SA, Smith GC, Boulton SJ, Tijsterman M | title = Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways | journal = Cell Reports | volume = 42 | issue = 2 | pages = 112019 | date = February 2023 | pmid = 36701230 | doi = 10.1016/j.celrep.2023.112019 | s2cid = 256273893 | doi-access = free | author9 = Smith GCM | hdl = 1887/3753233 | hdl-access = free }}{{cite journal | vauthors = Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL | title = Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining | journal = Nature Biotechnology | volume = 33 | issue = 5 | pages = 538–542 | date = May 2015 | pmid = 25798939 | pmc = 4618510 | doi = 10.1038/nbt.3190 }} A system with the Cpf1 effector protein was created that is induced by small molecules VE-822 and AZD-7762.{{cite journal | vauthors = Ma X, Chen X, Jin Y, Ge W, Wang W, Kong L, Ji J, Guo X, Huang J, Feng XH, Fu J, Zhu S | title = Small molecules promote CRISPR-Cpf1-mediated genome editing in human pluripotent stem cells | journal = Nature Communications | volume = 9 | issue = 1 | pages = 1303 | date = April 2018 | pmid = 29610531 | pmc = 5880812 | doi = 10.1038/s41467-018-03760-5 | bibcode = 2018NatCo...9.1303M }} These systems allow conditional control of CRISPR activity for improved precision, efficiency, and spatiotemporal control. Spatiotemporal control is a form of removing off-target effects—only certain cells or parts of the organism may need to be modified, and thus light or small molecules can be used as a way to conduct this. Efficiency of the CRISPR-Cas9 system is also greatly increased by proper delivery of the DNA instructions for creating the proteins and necessary reagents.

CRISPR also utilizes single base-pair editing proteins to create specific edits at one or two bases in the target sequence. CRISPR/Cas9 was fused with specific enzymes that initially could only change C to T and G to A mutations and their reverse. This was accomplished eventually without requiring any DNA cleavage.{{cite journal | vauthors = Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A | title = Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems | journal = Science | volume = 353 | issue = 6305 | pages = aaf8729 | date = September 2016 | pmid = 27492474 | doi = 10.1126/science.aaf8729 | s2cid = 5122081 }}{{cite journal | vauthors = Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR | title = Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage | journal = Nature | volume = 533 | issue = 7603 | pages = 420–424 | date = May 2016 | pmid = 27096365 | pmc = 4873371 | doi = 10.1038/nature17946 | bibcode = 2016Natur.533..420K }}{{cite journal | vauthors = Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR | title = Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage | journal = Nature | volume = 551 | issue = 7681 | pages = 464–471 | date = November 2017 | pmid = 29160308 | pmc = 5726555 | doi = 10.1038/nature24644 | bibcode = 2017Natur.551..464G }} With the fusion of another enzyme, the base editing CRISPR-Cas9 system can also edit C to G and its reverse.{{cite journal | vauthors = Chen L, Park JE, Paa P, Rajakumar PD, Prekop HT, Chew YT, Manivannan SN, Chew WL | title = Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins | journal = Nature Communications | volume = 12 | issue = 1 | pages = 1384 | date = March 2021 | pmid = 33654077 | pmc = 7925527 | doi = 10.1038/s41467-021-21559-9 | bibcode = 2021NatCo..12.1384C }}

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a gene-editing technology that can induce double-strand breaks (DSBs) anywhere guide ribonucleic acids (gRNA) can bind with the protospacer adjacent motif (PAM) sequence. Single-strand nicks can also be induced by Cas9 active-site mutants,{{cite journal | vauthors = Gasiunas G, Barrangou R, Horvath P, Siksnys V | title = Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 109 | issue = 39 | pages = E2579–E2586 | date = September 2012 | pmid = 22949671 | pmc = 3465414 | doi = 10.1073/pnas.1208507109 | doi-access = free }} also known as Cas9 nickases.{{cite journal | vauthors = Satomura A, Nishioka R, Mori H, Sato K, Kuroda K, Ueda M | title = Precise genome-wide base editing by the CRISPR Nickase system in yeast | journal = Scientific Reports | volume = 7 | issue = 1 | pages = 2095 | date = May 2017 | pmid = 28522803 | pmc = 5437071 | doi = 10.1038/s41598-017-02013-7 | bibcode = 2017NatSR...7.2095S }} By simply changing the sequence of gRNA, the Cas9-endonuclease can be delivered to a gene of interest and induce DSBs.{{cite journal | vauthors = Hiranniramol K, Chen Y, Liu W, Wang X | title = Generalizable sgRNA design for improved CRISPR/Cas9 editing efficiency | journal = Bioinformatics | volume = 36 | issue = 9 | pages = 2684–2689 | date = May 2020 | pmid = 31971562 | pmc = 7203743 | doi = 10.1093/bioinformatics/btaa041 }} The efficiency of Cas9-endonuclease and the ease by which genes can be targeted led to the development of CRISPR-knockout (KO) libraries both for mouse and human cells, which can cover either specific gene sets of interest or the whole-genome.{{cite journal | vauthors = Agrotis A, Ketteler R | title = A new age in functional genomics using CRISPR/Cas9 in arrayed library screening | journal = Frontiers in Genetics | volume = 6 | pages = 300 | date = 2015-09-24 | pmid = 26442115 | pmc = 4585242 | doi = 10.3389/fgene.2015.00300 | doi-access = free }}{{cite journal | vauthors = Yu JS, Yusa K | title = Genome-wide CRISPR-Cas9 screening in mammalian cells | journal = Methods | volume = 164-165 | pages = 29–35 | date = July 2019 | pmid = 31034882 | doi = 10.1016/j.ymeth.2019.04.015 | s2cid = 140275157 }} CRISPR screening helps scientists to create a systematic and high-throughput genetic perturbation within live model organisms. This genetic perturbation is necessary for fully understanding gene function and epigenetic regulation.{{cite journal | vauthors = Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham MD, Sanjana NE, Zhang F | title = Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening | journal = Nature Protocols | volume = 12 | issue = 4 | pages = 828–863 | date = April 2017 | pmid = 28333914 | pmc = 5526071 | doi = 10.1038/nprot.2017.016 }} The advantage of pooled CRISPR libraries is that more genes can be targeted at once.{{cn|date=April 2025}}

Knock-out libraries are created in a way to achieve equal representation and performance across all expressed gRNAs and carry an antibiotic or fluorescent selection marker that can be used to recover transduced cells.{{cite journal | vauthors = Kurata M, Yamamoto K, Moriarity BS, Kitagawa M, Largaespada DA | title = CRISPR/Cas9 library screening for drug target discovery | journal = Journal of Human Genetics | volume = 63 | issue = 2 | pages = 179–186 | date = February 2018 | pmid = 29158600 | doi = 10.1038/s10038-017-0376-9 | s2cid = 3308058 }} There are two plasmid systems in CRISPR/Cas9 libraries. First, is all in one plasmid, where sgRNA and Cas9 are produced simultaneously in a transfected cell. Second, is a two-vector system: sgRNA and Cas9 plasmids are delivered separately. It is important to deliver thousands of unique sgRNAs-containing vectors to a single vessel of cells by viral transduction at low multiplicity of infection (MOI, typically at 0.1–0.6), it prevents the probability that an individual cell clone will get more than one type of sgRNA otherwise it can lead to incorrect assignment of genotype to phenotype.

Once a pooled library is prepared it is necessary to carry out a deep sequencing (NGS, next generation sequencing) of PCR-amplified plasmid DNA in order to reveal abundance of sgRNAs. Cells of interest can be consequentially infected by the library and then selected according to the phenotype. There are 2 types of selection: negative and positive. By negative selection dead or slow growing cells are efficiently detected. It can identify survival-essential genes, which can further serve as candidates for molecularly targeted drugs. On the other hand, positive selection gives a collection of growth-advantage acquired populations by random mutagenesis. After selection genomic DNA is collected and sequenced by NGS. Depletion or enrichment of sgRNAs is detected and compared to the original sgRNA library, annotated with the target gene that sgRNA corresponds to. Statistical analysis then identifies genes that are significantly likely to be relevant to the phenotype of interest.

Apart from knock-out there are also knock-down (CRISPRi) and activation (CRISPRa) libraries, which use the ability of proteolytically deactivated Cas9-fusion proteins (dCas9) to bind target DNA, which means that a gene of interest is not cut but is over-expressed or repressed. It made CRISPR/Cas9 system even more interesting in gene editing. Inactive dCas9 protein modulate gene expression by targeting dCas9-repressors or activators toward promoter or transcriptional start sites of target genes. For repressing genes Cas9 can be fused to KRAB effector domain that makes complex with gRNA, whereas CRISPRa utilizes dCas9 fused to different transcriptional activation domains, which are further directed by gRNA to promoter regions to upregulate expression.{{cite journal | vauthors = McDade JR, Waxmonsky NC, Swanson LE, Fan M | title = Practical Considerations for Using Pooled Lentiviral CRISPR Libraries | journal = Current Protocols in Molecular Biology | volume = 115 | issue = 1 | pages = 31.5.1–31.5.13 | date = July 2016 | pmid = 27366891 | doi = 10.1002/cpmb.8 | s2cid = 5055878 }}{{cite journal | vauthors = Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan S, Shivalila CS, Dadon DB, Jaenisch R | title = Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system | journal = Cell Research | volume = 23 | issue = 10 | pages = 1163–1171 | date = October 2013 | pmid = 23979020 | pmc = 3790238 | doi = 10.1038/cr.2013.122 }}{{cite journal | vauthors = Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, Guimaraes C, Panning B, Ploegh HL, Bassik MC, Qi LS, Kampmann M, Weissman JS | title = Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation | journal = Cell | volume = 159 | issue = 3 | pages = 647–661 | date = October 2014 | pmid = 25307932 | pmc = 4253859 | doi = 10.1016/j.cell.2014.09.029 }}

Cas9 genomic modification has allowed for the quick and efficient generation of transgenic models within the field of genetics. Cas9 can be easily introduced into the target cells along with sgRNA via plasmid transfection in order to model the spread of diseases and the cell's response to and defense against infection.{{cite journal |vauthors=Dow LE |title=Modeling Disease In Vivo With CRISPR/Cas9 |journal=Trends in Molecular Medicine |volume=21 |issue = 10 |pages=609–621 |date=October 2015 |pmid=26432018 |pmc=4592741 |doi=10.1016/j.molmed.2015.07.006 }} The ability of Cas9 to be introduced in vivo allows for the creation of more accurate models of gene function and mutation effects, all while avoiding the off-target mutations typically observed with older methods of genetic engineering.{{cn|date=April 2025}}

The CRISPR and Cas9 revolution in genomic modeling does not extend only to mammals. Traditional genomic models such as Drosophila melanogaster, one of the first model organisms, have seen further refinement in their resolution with the use of Cas9. Cas9 uses cell-specific promoters allowing a controlled use of the Cas9. Cas9 is an accurate method of treating diseases due to the targeting of the Cas9 enzyme only affecting certain cell types. The cells undergoing the Cas9 therapy can also be removed and reintroduced to provide amplified effects of the therapy.{{Cite book |vauthors=Doudna J, Mali P |title=CRISPR-Cas: A Laboratory Manual |year=2016 |isbn=978-1-62182-130-4 |location=Cold Spring Harbor, New York |publisher=Cold Spring Harbor Laboratory Press |oclc=922914104}}

CRISPR-Cas9 can be used to edit the DNA of organisms in vivo and to eliminate individual genes or even entire chromosomes from an organism at any point in its development. Chromosomes that have been successfully deleted in vivo using CRISPR techniques include the Y chromosome and X chromosome of adult lab mice and human chromosomes 14 and 21, in embryonic stem cell lines and aneuploid mice respectively. This method might be useful for treating genetic disorders caused by abnormal numbers of chromosomes, such as Down syndrome and intersex disorders.{{cite journal |vauthors=Zuo E, Huo X, Yao X, Hu X, Sun Y, Yin J, He B, Wang X, Shi L, Ping J, Wei Y, Ying W, Wei W, Liu W, Tang C, Li Y, Hu J, Yang H |title=CRISPR/Cas9-mediated targeted chromosome elimination |journal=Genome Biology |volume=18 |issue =1 |pages=224 |date=November 2017 |pmid=29178945 |pmc=5701507 |doi=10.1186/s13059-017-1354-4 |doi-access=free }}

• {{cite web |date=Nov 27, 2017 |title=CRISPR Used to Eliminate Targeted Chromosomes in New Study |website=Genome Web |url=https://www.genomeweb.com/gene-silencinggene-editing/crispr-used-eliminate-targeted-chromosomes-new-study |url-access=registration}}

Successful in vivo genome editing using CRISPR-Cas9 has been shown in numerous model organisms, including Escherichia coli,{{cite journal |vauthors = Javed MR, Sadaf M, Ahmed T, Jamil A, Nawaz M, Abbas H, Ijaz A |title = CRISPR-Cas System: History and Prospects as a Genome Editing Tool in Microorganisms |journal = Current Microbiology |volume = 75 |issue = 12 |pages = 1675–1683 |date = December 2018 |pmid = 30078067 |doi = 10.1007/s00284-018-1547-4 |s2cid = 51920661 |department=review }} Saccharomyces cerevisiae,{{cite journal |vauthors=DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM |title=Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems |journal=Nucleic Acids Res |volume=41 |issue=7 |pages=4336–43 |date=April 2013 |pmid=23460208 |pmc=3627607 |doi=10.1093/nar/gkt135 |url=}}{{cite journal |vauthors = Giersch RM, Finnigan GC |title = Yeast Still a Beast: Diverse Applications of CRISPR/Cas Editing Technology in S. cerevisiae |journal=The Yale Journal of Biology and Medicine |volume =90 |issue=4 |pages=643–651 |date=December 2017 |pmid = 29259528 |pmc = 5733842 }} Candida albicans, Methanosarcina acetivorans,{{cite journal |vauthors = Dhamad AE, Lessner DJ |title = A CRISPRi-dCas9 System for Archaea and Its Use To Examine Gene Function during Nitrogen Fixation by Methanosarcina acetivorans |journal = Applied and Environmental Microbiology |volume = 86 |issue = 21 |pages = e01402–20 |date=October 2020 |pmid = 32826220 |pmc=7580536 |doi=10.1128/AEM.01402-20 |bibcode = 2020ApEnM..86E1402D |veditors= Atomi H }}{{cite journal |vauthors = Raschmanová H, Weninger A, Glieder A, Kovar K, Vogl T |title = Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: Current state and future prospects |journal=Biotechnology Advances |volume = 36 |issue = 3 |pages = 641–665 |date = 2018 |pmid = 29331410 |doi=10.1016/j.biotechadv.2018.01.006 |doi-access = free |department=review }} Caenorhabditis elegans, Arabidopsis spp.,{{cite journal |vauthors = Khurshid H, Jan SA, Shinwari ZK, Jamal M, Shah SH |title = An Era of CRISPR/ Cas9 Mediated Plant Genome Editing |journal = Current Issues in Molecular Biology |volume = 26 |pages = 47–54 |date = 2018 |pmid = 28879855 |doi = 10.21775/cimb.026.047 |department = review |doi-access = free }} Danio rerio,{{cite journal |vauthors = Simone BW, Martínez-Gálvez G, WareJoncas Z, Ekker SC |title = Fishing for understanding: Unlocking the zebrafish gene editor's toolbox |journal = Methods |volume = 150 |pages = 3–10 |date = November 2018 |pmid = 30076892 |pmc = 6590056 |doi = 10.1016/j.ymeth.2018.07.012 |department = review }} and the house mouse (mus musculus).{{cite journal |vauthors=Singh P, Schimenti JC, Bolcun-Filas E |title=A mouse generalist practical guide to CRISPR applications |journal=Genetics |volume=199 |issue=1 |pages=1–15 |date=January 2015 |pmid=25271304 |pmc=4286675 |doi=10.1534/genetics.114.169771 |department = review }}{{cite journal |vauthors = Soni D, Wang DM, Regmi SC, Mittal M, Vogel SM, Schlüter D, Tiruppathi C |title = Deubiquitinase function of A20 maintains and repairs endothelial barrier after lung vascular injury |journal = Cell Death Discovery |volume = 4 |issue = 60 |pages = 60 |date = May 2018 |pmid = 29796309 |pmc = 5955943 |doi = 10.1038/s41420-018-0056-3 }} Successes have been achieved in the study of basic biology, in the creation of disease models,{{cite journal |vauthors = Ma D, Liu F |title = Genome Editing and Its Applications in Model Organisms |journal=Genomics, Proteomics & Bioinformatics |volume = 13 |issue = 6 |pages = 336–344 |date = December 2015 |pmid = 26762955 |pmc = 4747648 |doi = 10.1016/j.gpb.2015.12.001 |department = review }}{{cite journal |vauthors = Birling MC, Herault Y, Pavlovic G |title = Modeling human disease in rodents by CRISPR/Cas9 genome editing |journal = Mammalian Genome |volume = 28 |issue = 7–8 |pages = 291–301 |date = August 2017 |pmid = 28677007 |pmc = 5569124 |doi = 10.1007/s00335-017-9703-x }} and in the experimental treatment of disease models.{{cite journal |vauthors = Gao X, Tao Y, Lamas V, Huang M, Yeh WH, Pan B, Hu YJ, Hu JH, Thompson DB, Shu Y, Li Y, Wang H, Yang S, Xu Q, Polley DB, Liberman MC, Kong WJ, Holt JR, Chen ZY, Liu DR |title = Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents |journal=Nature |volume=553 |issue=7687 |pages=217–221 |date=January 2018 |pmid = 29258297 |pmc=5784267 |doi = 10.1038/nature25164 |bibcode = 2018Natur.553..217G }}

Concerns have been raised that off-target effects (editing of genes besides the ones intended) may confound the results of a CRISPR gene editing experiment (i.e. the observed phenotype change may not be due to modifying the target gene, but some other gene). Modifications to CRISPR have been made to minimize the possibility of off-target effects. Orthogonal CRISPR experiments are often recommended to confirm the results of a gene editing experiment.{{cite journal |vauthors = Kadam US, Shelake RM, Chavhan RL, Suprasanna P |title = Concerns regarding 'off-target' activity of genome editing endonucleases |journal = Plant Physiology and Biochemistry |volume = 131 |pages = 22–30 |date = October 2018 |pmid = 29653762 |doi = 10.1016/j.plaphy.2018.03.027 |bibcode = 2018PlPB..131...22K |s2cid = 4846191 |department = review }}{{cite journal |vauthors = Kimberland ML, Hou W, Alfonso-Pecchio A, Wilson S, Rao Y, Zhang S, Lu Q |title = Strategies for controlling CRISPR/Cas9 off-target effects and biological variations in mammalian genome editing experiments |journal = Journal of Biotechnology |volume = 284 |pages = 91–101 |date = October 2018 |pmid = 30142414 |doi = 10.1016/j.jbiotec.2018.08.007 |s2cid = 52078796 |department = review }}

CRISPR simplifies the creation of genetically modified organisms for research which mimic disease or show what happens when a gene is knocked down or mutated. CRISPR may be used at the germline level to create organisms in which the targeted gene is changed everywhere (i.e. in all cells/tissues/organs of a multicellular organism), or it may be used in non-germline cells to create local changes that only affect certain cell populations within the organism.{{cite journal |vauthors = van Erp PB, Bloomer G, Wilkinson R, Wiedenheft B |title = The history and market impact of CRISPR RNA-guided nucleases |journal = Current Opinion in Virology |volume = 12 |pages = 85–90 |date = June 2015 |pmid = 25914022 |pmc = 4470805 |doi = 10.1016/j.coviro.2015.03.011 }}{{cite journal |vauthors = Maggio I, Gonçalves MA |title = Genome editing at the crossroads of delivery, specificity, and fidelity |journal = Trends in Biotechnology |volume = 33 |issue = 5 |pages = 280–291 |date = May 2015 |pmid = 25819765 |doi = 10.1016/j.tibtech.2015.02.011 |doi-access = free }}{{cite journal |vauthors = Rath D, Amlinger L, Rath A, Lundgren M |title=The CRISPR-Cas immune system: biology, mechanisms and applications |journal=Biochimie |volume = 117 |pages=119–128 |date=October 2015 |pmid = 25868999 |doi=10.1016/j.biochi.2015.03.025 |doi-access=free }}

CRISPR can be utilized to create human cellular models of disease.{{Cite web |url=https://www.livescience.tech/2020/02/06/what-is-crispr-how-does-it-work-is-it-gene-editing/ |title=What Is CRISPR? How Does It Work? Is It Gene Editing? |date=2018-04-30 |website=LiveScience.Tech |language=en-US|access-date=2020-02-06 |archive-date=2020-02-06 |archive-url=https://web.archive.org/web/20200206165950/https://www.livescience.tech/2020/02/06/what-is-crispr-how-does-it-work-is-it-gene-editing/ |url-status=dead}} For instance, when applied to human pluripotent stem cells, CRISPR has been used to introduce targeted mutations in genes relevant to polycystic kidney disease (PKD) and focal segmental glomerulosclerosis (FSGS).{{cite journal |vauthors = Freedman BS, Brooks CR, Lam AQ, Fu H, Morizane R, Agrawal V, Saad AF, Li MK, Hughes MR, Werff RV, Peters DT, Lu J, Baccei A, Siedlecki AM, Valerius MT, Musunuru K, McNagny KM, Steinman TI, Zhou J, Lerou PH, Bonventre JV |title = Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids |journal=Nature Communications |volume = 6 |pages = 8715 |date = October 2015 |pmid = 26493500 |pmc = 4620584 |doi = 10.1038/ncomms9715 |bibcode = 2015NatCo...6.8715F }} These CRISPR-modified pluripotent stem cells were subsequently grown into human kidney organoids that exhibited disease-specific phenotypes. Kidney organoids from stem cells with PKD mutations formed large, translucent cyst structures from kidney tubules. The cysts were capable of reaching macroscopic dimensions, up to one centimeter in diameter.{{cite journal |vauthors = Cruz NM, Song X, Czerniecki SM, Gulieva RE, Churchill AJ, Kim YK, Winston K, Tran LM, Diaz MA, Fu H, Finn LS, Pei Y, Himmelfarb J, Freedman BS |title = Organoid cystogenesis reveals a critical role of microenvironment in human polycystic kidney disease |journal = Nature Materials |volume = 16 |issue = 11 |pages = 1112–1119 |date = November 2017 |pmid = 28967916 |pmc = 5936694 |doi = 10.1038/nmat4994 |bibcode = 2017NatMa..16.1112C }} Kidney organoids with mutations in a gene linked to FSGS developed junctional defects between podocytes, the filtering cells affected in that disease. This was traced to the inability of podocytes to form microvilli between adjacent cells.{{cite journal |vauthors = Kim YK, Refaeli I, Brooks CR, Jing P, Gulieva RE, Hughes MR, Cruz NM, Liu Y, Churchill AJ, Wang Y, Fu H, Pippin JW, Lin LY, Shankland SJ, Vogl AW, McNagny KM, Freedman BS |title = Gene-Edited Human Kidney Organoids Reveal Mechanisms of Disease in Podocyte Development |journal = Stem Cells |volume = 35 |issue = 12 |pages = 2366–2378 |date = December 2017 |pmid = 28905451 |pmc = 5742857 |doi = 10.1002/stem.2707 }} Importantly, these disease phenotypes were absent in control organoids of identical genetic background, but lacking the CRISPR modifications.

A similar approach was taken to model long QT syndrome in cardiomyocytes derived from pluripotent stem cells.{{cite journal |vauthors = Bellin M, Casini S, Davis RP, D'Aniello C, Haas J, Ward-van Oostwaard D, Tertoolen LG, Jung CB, Elliott DA, Welling A, Laugwitz KL, Moretti A, Mummery CL |title = Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome |journal = The EMBO Journal |volume = 32 |issue = 24 |pages = 3161–3175 |date = December 2013 |pmid = 24213244 |pmc = 3981141 |doi = 10.1038/emboj.2013.240 }} These CRISPR-generated cellular models, with isogenic controls, provide a new way to study human disease and test drugs.

CRISPR-Cas technology has been proposed as a treatment for multiple human diseases, especially those with a genetic cause.{{cite journal | vauthors = Cai L, Fisher AL, Huang H, Xie Z | title = CRISPR-mediated genome editing and human diseases | journal = Genes & Diseases | volume = 3 | issue = 4 | pages = 244–251 | date = December 2016 | pmid = 30258895 | pmc = 6150104 | doi = 10.1016/j.gendis.2016.07.003 }} Its ability to modify specific DNA sequences makes it a tool with potential to fix disease-causing mutations. Early research in animal models suggest that therapies based on CRISPR technology have potential to treat a wide range of diseases,{{Cite news|url=https://labiotech.eu/tops/crispr-technology-cure-disease/|title=Seven Diseases That CRISPR Technology Could Cure|date=2018-06-25|work=Labiotech.eu|access-date=2018-08-22 }} including cancer,{{Cite web|url=https://immuno-oncologynews.com/crisprcas9-and-cancer/|title=CRISPR/Cas9 and Cancer|website=Immuno-Oncology News|language=en-US|access-date=2019-02-18|date=2018-04-27}} progeria,{{Cite web| vauthors = Crossley M |title=New CRISPR technology could revolutionise gene therapy, offering new hope to people with genetic diseases|url=http://theconversation.com/new-crispr-technology-could-revolutionise-gene-therapy-offering-new-hope-to-people-with-genetic-diseases-153641|access-date=2021-02-03|website=The Conversation|date=February 2021 |language=en}} beta-thalassemia,{{cite journal | vauthors = Cromer MK, Camarena J, Martin RM, Lesch BJ, Vakulskas CA, Bode NM, Kurgan G, Collingwood MA, Rettig GR, Behlke MA, Lemgart VT, Zhang Y, Goyal A, Zhao F, Ponce E, Srifa W, Bak RO, Uchida N, Majeti R, Sheehan VA, Tisdale JF, Dever DP, Porteus MH | title = Gene replacement of α-globin with β-globin restores hemoglobin balance in β-thalassemia-derived hematopoietic stem and progenitor cells | journal = Nature Medicine | volume = 27 | issue = 4 | pages = 677–687 | date = April 2021 | pmid = 33737751 | pmc = 8265212 | doi = 10.1038/s41591-021-01284-y }}{{cite journal | vauthors = Xie F, Ye L, Chang JC, Beyer AI, Wang J, Muench MO, Kan YW | title = Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac | journal = Genome Research | volume = 24 | issue = 9 | pages = 1526–1533 | date = September 2014 | pmid = 25096406 | pmc = 4158758 | doi = 10.1101/gr.173427.114 }}{{cite journal | vauthors = Frangoul H, Altshuler D, Cappellini MD, Chen YS, Domm J, Eustace BK, Foell J, de la Fuente J, Grupp S, Handgretinger R, Ho TW, Kattamis A, Kernytsky A, Lekstrom-Himes J, Li AM, Locatelli F, Mapara MY, de Montalembert M, Rondelli D, Sharma A, Sheth S, Soni S, Steinberg MH, Wall D, Yen A, Corbacioglu S | title = CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia | journal = The New England Journal of Medicine | volume = 384 | issue = 3 | pages = 252–260 | date = January 2021 | pmid = 33283989 | doi = 10.1056/NEJMoa2031054 | s2cid = 227521558 | doi-access = free }} sickle cell disease,{{cite journal | vauthors = Dever DP, Bak RO, Reinisch A, Camarena J, Washington G, Nicolas CE, Pavel-Dinu M, Saxena N, Wilkens AB, Mantri S, Uchida N, Hendel A, Narla A, Majeti R, Weinberg KI, Porteus MH | title = CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells | journal = Nature | volume = 539 | issue = 7629 | pages = 384–389 | date = November 2016 | pmid = 27820943 | pmc = 5898607 | doi = 10.1038/nature20134 | bibcode = 2016Natur.539..384D }} hemophilia,{{Cite web|url=https://www.genengnews.com/gen-news-highlights/crispr-one-shot-cell-therapy-for-hemophilia-developed/81255772|title=CRISPR 'One Shot' Cell Therapy for Hemophilia Developed |website=GEN |access-date=2018-08-22|date=2018-05-02}} cystic fibrosis,{{cite journal | vauthors = Marangi M, Pistritto G | title = Innovative Therapeutic Strategies for Cystic Fibrosis: Moving Forward to CRISPR Technique | journal = Frontiers in Pharmacology | volume = 9 | pages = 396 | date = 2018-04-20 | pmid = 29731717 | pmc = 5920621 | doi = 10.3389/fphar.2018.00396 | doi-access = free }} Duchenne's muscular dystrophy,{{cite journal | vauthors = Bengtsson NE, Hall JK, Odom GL, Phelps MP, Andrus CR, Hawkins RD, Hauschka SD, Chamberlain JR, Chamberlain JS | title = Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy | journal = Nature Communications | volume = 8 | pages = 14454 | date = February 2017 | pmid = 28195574 | pmc = 5316861 | doi = 10.1038/ncomms14454 | bibcode = 2017NatCo...814454B }} Huntington's disease,{{cite journal | vauthors = Eisenstein M | title = CRISPR takes on Huntington's disease | journal = Nature | volume = 557 | issue = 7707 | pages = S42–S43 | date = May 2018 | pmid = 29844549 | doi = 10.1038/d41586-018-05177-y | doi-access = free | bibcode = 2018Natur.557S..42E }}{{cite journal | vauthors = Dabrowska M, Juzwa W, Krzyzosiak WJ, Olejniczak M | title = Precise Excision of the CAG Tract from the Huntingtin Gene by Cas9 Nickases | journal = Frontiers in Neuroscience | volume = 12 | pages = 75 | date = 2018 | pmid = 29535594 | pmc = 5834764 | doi = 10.3389/fnins.2018.00075 | doi-access = free }} transthyretin amyloidosis and heart disease.{{cite journal | vauthors = King A | title = A CRISPR edit for heart disease | journal = Nature | volume = 555 | issue = 7695 | pages = S23–S25 | date = March 2018 | pmid = 29517035 | doi = 10.1038/d41586-018-02482-4 | doi-access = free | bibcode = 2018Natur.555.....K }} CRISPR has also been used to cure malaria in mosquitos, which could eliminate the vector and the disease in humans.{{cite journal | vauthors = Scudellari M | title = Self-destructing mosquitoes and sterilized rodents: the promise of gene drives | journal = Nature | volume = 571 | issue = 7764 | pages = 160–162 | date = July 2019 | pmid = 31289403 | doi = 10.1038/d41586-019-02087-5 | doi-access = free | bibcode = 2019Natur.571..160S }} CRISPR may also have applications in tissue engineering and regenerative medicine, such as by creating human blood vessels that lack expression of MHC class II proteins, which often cause transplant rejection.{{cite journal | vauthors = Abrahimi P, Chang WG, Kluger MS, Qyang Y, Tellides G, Saltzman WM, Pober JS | title = Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9 | journal = Circulation Research | volume = 117 | issue = 2 | pages = 121–128 | date = July 2015 | pmid = 25940550 | pmc = 4490936 | doi = 10.1161/CIRCRESAHA.117.306290 }}

In addition, clinical trials to cure beta thalassemia and sickle cell disease in human patients using CRISPR-Cas9 technology have shown promising results.{{Cite news|title=A Year In, 1st Patient To Get Gene Editing For Sickle Cell Disease Is Thriving|url=https://www.npr.org/sections/health-shots/2020/06/23/877543610/a-year-in-1st-patient-to-get-gene-editing-for-sickle-cell-disease-is-thriving|access-date=2021-02-03|website=NPR.org|language=en}}{{Cite web|title=CRISPR technology to cure sickle cell disease|url=https://www.sciencedaily.com/releases/2021/01/210121131904.htm|access-date=2021-02-03|website=ScienceDaily|language=en}} In December 2023, the US Food and Drug Administration (FDA) approved the first cell-based gene therapies for treating sickle cell disease, Casgevy and Lyfgenia. Casgevy is the first FDA approved gene therapy to use the CRISPR-Cas9 technology and works by modifying a patient's hematopoietic stem cells.{{Cite web | author = Office of the Commissioner |date=2024-08-09 |title=FDA Approves First Gene Therapies to Treat Patients with Sickle Cell Disease |url=https://www.fda.gov/news-events/press-announcements/fda-approves-first-gene-therapies-treat-patients-sickle-cell-disease |access-date=2025-01-08 |website=FDA |language=en}}

Nevertheless, there remains a few limitations of the technology's use in gene therapy: the relatively high frequency of off-target effect, the requirement for a PAM sequence near the target site, p53 mediated apoptosis by CRISPR-induced double-strand breaks and immunogenic toxicity due to the delivery system typically by virus.{{cite journal | vauthors = Uddin F, Rudin CM, Sen T | title = CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future | journal = Frontiers in Oncology | volume = 10 | pages = 1387 | date = August 2020 | pmid = 32850447 | pmc = 7427626 | doi = 10.3389/fonc.2020.01387 | doi-access = free }}

CRISPR has also found many applications in developing cell-based immunotherapies.{{cite journal | vauthors = Jensen TI, Axelgaard E, Bak RO | title = Therapeutic gene editing in haematological disorders with CRISPR/Cas9 | journal = British Journal of Haematology | volume = 185 | issue = 5 | pages = 821–835 | date = June 2019 | pmid = 30864164 | doi = 10.1111/bjh.15851 | s2cid = 76663873 | doi-access = free }} The first clinical trial involving CRISPR started in 2016. It involved taking immune cells from people with lung cancer, using CRISPR to edit out the gene which expressed Programmed cell death protein 1 (PD-1), then administering the altered cells back to the same person. 20 other trials were under way or nearly ready, mostly in China, {{as of|2017|lc=y}}.

In 2016, the United States Food and Drug Administration (FDA) approved a clinical trial in which CRISPR would be used to alter T cells extracted from people with different kinds of cancer and then administer those engineered T cells back to the same people.{{cite journal |doi=10.1038/nature.2016.20137 |title=First CRISPR clinical trial gets green light from US panel |journal=Nature |year=2016 | vauthors = Reardon S |s2cid=89466280 }}

In November 2020, in mouse animal models, CRISPR was used effectively to treat glioblastoma (fast-growing brain tumor) and metastatic ovarian cancer, as those are two cancers with some of the worst best-case prognosis and are typically diagnosed during their later stages. The treatments have resulted in inhibited tumor growth, and increased survival by 80% for metastatic ovarian cancer and tumor cell apoptosis, inhibited tumor growth by 50%, and improved survival by 30% for glioblastoma.{{cite journal | vauthors = Rosenblum D, Gutkin A, Kedmi R, Ramishetti S, Veiga N, Jacobi AM, Schubert MS, Friedmann-Morvinski D, Cohen ZR, Behlke MA, Lieberman J, Peer D | title = CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy | journal = Science Advances | volume = 6 | issue = 47 | pages = eabc9450 | date = November 2020 | pmid = 33208369 | pmc = 7673804 | doi = 10.1126/sciadv.abc9450 | doi-access = free | bibcode = 2020SciA....6.9450R }}

In October 2021, CRISPR Therapeutics announced results from their ongoing US-based Phase 1 trial for an allogeneic T cell therapy. These cells are sourced from healthy donors and are edited to attack cancer cells and avoid being seen as a threat by the recipient's immune system, and then multiplied into huge batches which can be given to large numbers of recipients.{{Cite web |title=CRISPR Clinical Trials: A 2022 Update |url=https://innovativegenomics.org/news/crispr-clinical-trials-2022/ |access-date=2022-05-02 |website=Innovative Genomics Institute (IGI) |date=29 March 2022 |language=en-US}}

In December 2022, a 13-year British girl that had been diagnosed with incurable T-Cell Acute Lymphoblastic Leukaemia was cured by doctors at Great Ormond Street Hospital, in the first documented use of therapeutic gene editing for this purpose, after undergoing six months of an experimental treatment, where previous attempts of other treatments failed. The procedure included reprogramming a healthy T-Cell to destroy the cancerous T-Cells to first rid her of Leukaemia, and then rebuilding her immune system from scratch using healthy immune cells.{{cite news| title=Base editing: Revolutionary therapy clears girl's incurable cancer | website=BBC News | date=11 December 2022 | url=https://www.bbc.co.uk/news/health-63859184 | access-date = 11 December 2022}} The team used BASE editing and had previously treated a case of acute lymphoblastic leukaemia in 2015 using TALENs.{{Cite news |date=2015-11-05 |title='Designer cells' reverse one-year-old's cancer |language=en-GB |work=BBC News |url=https://www.bbc.com/news/health-34731498 |access-date=2022-12-11}}

Type 1 Diabetes is an endocrine disorder which results from a lack of pancreatic beta cells to produce insulin, a vital compound in transporting blood sugar to cells for producing energy. Researchers have been trying to transplant healthy beta cells. CRISPR is used to edit the cells in order to reduce the chance the patient's body will reject the transplant.{{cn|date=April 2025}}

On November 17, 2021 CRISPR therapeutics and ViaCyte announced that the Canadian medical agency had approved their request for a clinical trial for VCTX210, a CRISPR-edited stem cell therapy designed to treat type 1 diabetes. This was significant because it was the first ever gene-edited therapy for diabetes that approached clinics. The same companies also developed a novel treatment for type 1 diabetes to produce insulin via a small medical implant that uses millions of pancreatic cells derived from CRISPR gene-edited stem cells.{{Cite web | vauthors = Lee LX |date=2022-05-12 |title=B.C. researchers launching clinical trial for first genetically engineered stem cell-based therapy for type 1 diabetes |url=https://news.ubc.ca/2022/05/12/b-c-researchers-launching-clinical-trial-for-first-genetically-engineered-stem-cell-based-therapy-for-type-1-diabetes/ |access-date=2023-12-08 |website=UBC News |language=en-US}}

In February 2022, a phase 1 trial was conducted in which one patient volunteer received treatment.{{Cite web |title=CRISPR Therapeutics and ViaCyte, Inc. Announce First Patient Dosed in... |url=http://www.crisprtx.com/about-us/press-releases-and-presentations/crispr-therapeutics-and-viacyte-inc-announce-first-patient-dosed-in-phase-1-clinical-trial-of-novel-gene-edited-cell-replacement-therapy-for-treatment-of-type-1-diabetes-t1d |access-date=2022-05-02 |website=CRISPR |language=en}}

Human immunodeficiency virus (HIV) is a virus that attacks the body's immune system. While effective treatments exist which can allow patients to live healthy lives, HIV is retroactive meaning that it embeds an inactive version of itself in the human genome. CRISPR can be used to selectively remove the virus from the genome by designing guide RNA to target the retroactive HIV genome. One issue with this approach is that it requires the removal of the HIV genome from almost all cells, which can be difficult to realistically achieve.

Initial results in the treatment and cure of HIV have been rather successful, in 2021 9 out of 23 humanized mice treated with a combination of anti-retrovirals and CRISPR/Cas-9 had the virus become undetectable, even after the usual rebound period. None of the two treatments alone had such an effect.{{Cite web |author=National Institute on Drug Abuse |date=2020-02-14 |title=Antiretroviral Therapy Combined With CRISPR Gene Editing Can Eliminate HIV Infection in Mice |url=https://nida.nih.gov/news-events/nida-notes/2020/02/antiretroviral-therapy-combined-crispr-gene-editing-can-eliminate-hiv-infection-in-mice |access-date=2022-04-18 |website=National Institute on Drug Abuse |language=en}} Clinical trials in humans of a CRISPR–Cas9 based therapy, EBT-101 started in 2022.{{Cite web |title=First Clinical Trial of CRISPR-Based HIV Therapy Founded on Breakthrough Research at Lewis Katz School of Medicine {{!}} Lewis Katz School of Medicine at Temple University |url=https://medicine.temple.edu/news/first-clinical-trial-crispr-based-hiv-therapy-founded-breakthrough-research-lewis-katz-school |access-date=2022-04-18 |website=medicine.temple.edu |language=en}}{{Cite web |author=Excision BioTherapeutics |date=2022-03-24 |title=A Phase 1/2a, Sequential Cohort, Single Ascending Dose Study of the Safety, Tolerability, Biodistribution, and Pharmacodynamics of EBT 101 in Aviremic HIV-1 Infected Adults on Stable Antiretroviral Therapy |url=https://clinicaltrials.gov/ct2/show/NCT05144386}} In October 2023 an early-stage study on 3 people of EBT-101 reported that the treatment appeared to be safe with no major side effects but no data on its effectiveness was disclosed.{{Cite web |title=Three people were gene-edited in an effort to cure their HIV. The result is unknown. |url=https://www.technologyreview.com/2023/10/25/1082306/gene-editing-crispr-hiv-experiment/ |access-date=2024-03-20 |website=MIT Technology Review |language=en}} In March 2024 another CRISPR therapy from researchers of the university of Amsterdam reported the elimination of HIV in cell cultures.{{Cite web |title=HIV in cell culture can be completely eliminated using CRISPR-Cas gene editing technology, increasing hopes of cure |url=https://www.eurekalert.org/news-releases/1038161 |access-date=2024-03-20 |website=EurekAlert! |language=en}}{{Cite news |date=2024-03-20 |title=Scientists say they can cut HIV out of cells |url=https://www.bbc.com/news/health-68609297 |access-date=2024-03-20 |language=en-GB}}

CRISPR-Cas-based "RNA-guided nucleases" can be used to target virulence factors, genes encoding antibiotic resistance, and other medically relevant sequences of interest. This technology thus represents a novel form of antimicrobial therapy and a strategy by which to manipulate bacterial populations.{{cite journal | vauthors = Gomaa AA, Klumpe HE, Luo ML, Selle K, Barrangou R, Beisel CL | title = Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems | journal = mBio | volume = 5 | issue = 1 | pages = e00928–e00913 | date = January 2014 | pmid = 24473129 | pmc = 3903277 | doi = 10.1128/mBio.00928-13 }}{{cite journal | vauthors = Citorik RJ, Mimee M, Lu TK | title = Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases | journal = Nature Biotechnology | volume = 32 | issue = 11 | pages = 1141–1145 | date = November 2014 | pmid = 25240928 | pmc = 4237163 | doi = 10.1038/nbt.3011 | hdl = 1721.1/100834 }} Recent studies suggest a correlation between the interfering of the CRISPR-Cas locus and acquisition of antibiotic resistance.{{cite journal | vauthors = Gholizadeh P, Aghazadeh M, Asgharzadeh M, Kafil HS | title = Suppressing the CRISPR/Cas adaptive immune system in bacterial infections | journal = European Journal of Clinical Microbiology & Infectious Diseases | volume = 36 | issue = 11 | pages = 2043–2051 | date = November 2017 | pmid = 28601970 | doi = 10.1007/s10096-017-3036-2 | s2cid = 22716314 }} This system provides protection of bacteria against invading foreign DNA, such as transposons, bacteriophages, and plasmids. This system was shown to be a strong selective pressure for the acquisition of antibiotic resistance and virulence factor in bacterial pathogens.

Therapies based on CRISPR–Cas3 gene editing technology delivered by engineered bacteriophages could be used to destroy targeted DNA in pathogens.{{cite journal | vauthors = Gibney E | title = What to expect in 2018: science in the new year | journal = Nature | volume = 553 | issue = 7686 | pages = 12–13 | date = January 2018 | pmid = 29300040 | doi = 10.1038/d41586-018-00009-5 | doi-access = free | bibcode = 2018Natur.553...12G }} Cas3 is more destructive than the better known Cas9.{{cite news | vauthors = Taylor P |title=J&J takes stake in Locus' CRISPR-based 'Pac-Man' antimicrobials |url=https://www.fiercebiotech.com/biotech/j-j-takes-stake-locus-crispr-based-pac-man-antimicrobials |access-date=27 February 2019 |agency=Fierce Biotech |date=Jan 3, 2019}}{{cite journal | vauthors = Reardon S | title = Modified viruses deliver death to antibiotic-resistant bacteria | journal = Nature | volume = 546 | issue = 7660 | pages = 586–587 | date = June 2017 | pmid = 28661508 | doi = 10.1038/nature.2017.22173 | doi-access = free | bibcode = 2017Natur.546..586R }}

Research suggests that CRISPR is an effective way to limit replication of multiple herpesviruses. It was able to eradicate viral DNA in the case of Epstein–Barr virus (EBV). Anti-herpesvirus CRISPRs have promising applications such as removing cancer-causing EBV from tumor cells, helping rid donated organs for immunocompromised patients of viral invaders, or preventing cold sore outbreaks and recurrent eye infections by blocking HSV-1 reactivation. {{as of|2016|August}}, these were awaiting testing.{{cite journal | vauthors = van Diemen FR, Kruse EM, Hooykaas MJ, Bruggeling CE, Schürch AC, van Ham PM, Imhof SM, Nijhuis M, Wiertz EJ, Lebbink RJ | title = CRISPR/Cas9-Mediated Genome Editing of Herpesviruses Limits Productive and Latent Infections | journal = PLOS Pathogens | volume = 12 | issue = 6 | pages = e1005701 | date = June 2016 | pmid = 27362483 | pmc = 4928872 | doi = 10.1371/journal.ppat.1005701 | doi-access = free }}

• {{cite web |date=August 4, 2016 |title=Using CRISPR to combat viral infections: a new way to treat herpes? |work=PLOS Media |url=https://www.youtube.com/watch?v=lQaWh8VLkiU |via=YouTube}}

CRISPR may revive the concept of transplanting animal organs into people. Retroviruses present in animal genomes could harm transplant recipients. In 2015, a team eliminated 62 copies of a particular retroviral DNA sequence from the pig genome in a kidney epithelial cell. Researchers recently demonstrated the ability to birth live pig specimens after removing these retroviruses from their genome using CRISPR for the first time.{{Cite news|url=https://www.technologyreview.com/s/608579/crispr-opens-up-new-possibilities-for-transplants-using-pig-organs/|title=Using CRISPR on pigs could make their organs safer for human transplant| vauthors = Mullin E |work=MIT Technology Review|access-date=2017-09-09}}

CRISPR can be used to suppress gain of function mutations and to repair loss of function mutations in neurological disorders.{{cite journal | vauthors = Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, Sur M, Zhang F | title = In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9 | journal = Nature Biotechnology | volume = 33 | issue = 1 | pages = 102–106 | date = January 2015 | pmid = 25326897 | pmc = 4492112 | doi = 10.1038/nbt.3055 }} The gene editing tool has become a foothold in vivo application for assimilation of molecular pathways.{{cn|date=April 2025}}

CRISPR is unique to the development of solving neurological diseases for several reasons. For example, CRISPR allows researchers to quickly generate animal and human cell models, allowing them to study how genes function in a nervous system. By introducing mutations that pertain to various diseases within these cells, researchers can study the effects of the changes on nervous system development, function, and behavior.{{Cite web | vauthors = Feijo S |title=How Brown neuroscientists are using CRISPR to accelerate brain research — and more |url=https://www.brown.edu/news/2021-04-22/crispr-neuroscience |access-date=2023-12-08 |website=Brown University |language=en}} They can uncover the molecular mechanisms that contribute to these disorders, which is essential for developing effective treatments. This is particularly useful in modeling and treating complex neurological disorders such as Alzheimer's, Parkinson's, and epilepsy among others.{{cn|date=April 2025}}

Alzheimer's Disease (AD) is a neurodegenerative disease categorized by neuron loss and an accumulation of intracellular neurofibrillary tangles and extracellular amyloid plaques in the brain.{{cite journal | vauthors = De Plano LM, Calabrese G, Conoci S, Guglielmino SP, Oddo S, Caccamo A | title = Applications of CRISPR-Cas9 in Alzheimer's Disease and Related Disorders | journal = International Journal of Molecular Sciences | volume = 23 | issue = 15 | pages = 8714 | date = August 2022 | pmid = 35955847 | pmc = 9368966 | doi = 10.3390/ijms23158714 | doi-access = free }} Three pathogenic genes that cause early onset AD in humans have been identified, specifically amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2). Over 300 mutations have been detected in these genes, resulting in an increase in total β-amyloid (Aβ), Aβ42/40 ratio, and/or Aβ polymerization.

CRISPR has been used to correct for the dystrophin gene, which is responsible for Duchenne muscular dystrophy, and for the SCN1A mutation responsible for the epilepsy disorder Dravet syndrome.{{cite journal | vauthors = Erkut E, Yokota T | title = CRISPR Therapeutics for Duchenne Muscular Dystrophy | journal = International Journal of Molecular Sciences | volume = 23 | issue = 3 | pages = 1832 | date = February 2022 | pmid = 35163754 | pmc = 8836469 | doi = 10.3390/ijms23031832 | doi-access = free }}{{cite journal | vauthors = Yamagata T, Raveau M, Kobayashi K, Miyamoto H, Tatsukawa T, Ogiwara I, Itohara S, Hensch TK, Yamakawa K | title = CRISPR/dCas9-based Scn1a gene activation in inhibitory neurons ameliorates epileptic and behavioral phenotypes of Dravet syndrome model mice | journal = Neurobiology of Disease | volume = 141 | pages = 104954 | date = July 2020 | pmid = 32445790 | doi = 10.1016/j.nbd.2020.104954 | s2cid = 218762990 | doi-access = free }} A challenge of using CRISPR for neurological treatment is transferring its components across the blood-brain barrier. However, recent advancements in nanoparticle delivery systems and viral vectors have shown promise in overcoming this hurdle{{citation needed|date=September 2024}}. Looking to the future, the use of CRISPR in neuroscience is expected to increase as technology evolves.

The most commonly occurring worldwide eye diseases are cataract and retinitis pigmentosa (RP). These are caused by a missense mutation in the alpha chain that leads to permanent blindness. A challenge to the use of CRISPR in eye disease is that retinal tissue in the eye is free from body immune response. Researchers' approach for using CRISPR is to bag the gene coding retinal protein and edit the genome.{{cite journal | vauthors = Artero-Castro A, Long K, Bassett A, Ávila-Fernandez A, Cortón M, Vidal-Puig A, Jendelova P, Rodriguez-Jimenez FJ, Clemente E, Ayuso C, Erceg S | title = Gene Correction Recovers Phagocytosis in Retinal Pigment Epithelium Derived from Retinitis Pigmentosa-Human-Induced Pluripotent Stem Cells | journal = International Journal of Molecular Sciences | volume = 22 | issue = 4 | page = 2092 | date = February 2021 | pmid = 33672445 | pmc = 7923278 | doi = 10.3390/ijms22042092 | doi-access = free }}

The CRISPR treatment for LCA10 (the most common variant of Leber congenital amaurosis which is the leading cause of inherited childhood blindness) modifies the patient's defective photoreceptor gene.{{cn|date=April 2025}}

In March 2020, the first patient volunteer in this US-based study, sponsored by Editas Medicine, was given a low-dose of the treatment to test for safety. In June 2021, enrollment began for a high-dose adult and pediatric cohort of 4 patient volunteers each. Dosing of the new cohorts was expected to be completed by July 2022. In November 2022, Editas reported that 20% of the patients treated had significant improvements, but also announced that the resulting target population was too small to support continued independent development.{{cite web | url=https://www.biospace.com/article/despite-positive-response-editas-hits-pause-to-seek-partner-for-ocular-gene-program/ | title=Editas Hits Pause on LCA10 Program in Search of Partner | date=17 November 2022 }}

CRISPR technology has been shown to work efficiently in the treatment of heart disease. In the case of familial hypercholesterolemia (FH), cholesterol deposition in the walls of the artery causes blockage of blood flow. This is caused by mutation in low density lipoprotein cholesterol receptors (LDLC) which results in excessive release of cholesterol into the blood. This can be treated by deletion of a base pair in exon 4 of the LDLC receptor. This is a nonsense mutation. {{citation needed|date=September 2024}}

This disease comes under genetic disorders which are caused by mutation occurring in the structure of hemoglobin or due to substitution of different amino acids in globin chains. Due to this, the red blood cells (RBC) cause a string of obstacles such as heart failure, hindrance of blood vessels, defects in growth and optical problems.{{Cite journal |date=2017-09-12 |title=CRISPR-Cas9 Mediated Gene Editing: A Revolution in Genome Engineering |url=https://www.gavinpublishers.com/articles/short-commentary/Biomarkers-and-Applications/crispr-cas9-mediated-gene-editing-a-revolution-in-genome-engineering |journal=Biomarkers and Applications |volume=1 |issue=2 |doi=10.29011/2576-9588.100111 |doi-broken-date=2024-11-02 |doi-access=free |access-date=2023-12-10 |archive-date=2020-08-18 |archive-url=https://web.archive.org/web/20200818135521/https://www.gavinpublishers.com/articles/short-commentary/Biomarkers-and-Applications/crispr-cas9-mediated-gene-editing-a-revolution-in-genome-engineering }} To rehabilitate β-hemoglobinopathies, the patient's multipotent cells are transferred in a mice model to study the rate of gene therapy in ex-vivo which results in expression of mRNA and the gene being rectified. Intriguingly RBC half-life was also increased.{{cn|date=April 2025}}

Hemophilia is a loss of function in blood where clotting factors do not work properly. By using CRISPR-Cas9, a vector is inserted into bacteria.{{cite journal | vauthors = Huai C, Jia C, Sun R, Xu P, Min T, Wang Q, Zheng C, Chen H, Lu D | title = CRISPR/Cas9-mediated somatic and germline gene correction to restore hemostasis in hemophilia B mice | journal = Human Genetics | volume = 136 | issue = 7 | pages = 875–883 | date = July 2017 | pmid = 28508290 | doi = 10.1007/s00439-017-1801-z | s2cid = 253979773 }} The vector used is Adenoviral vector which helps in correction of genes.{{cn|date=April 2025}}

Successful CRISPR-Cas9 genome editing was first achieved in plants in August 2013.{{cite journal | vauthors = Feng Z, Zhang B, Ding W, Liu X, Yang DL, Wei P, Cao F, Zhu S, Zhang F, Mao Y, Zhu JK | title = Efficient genome editing in plants using a CRISPR/Cas system | journal = Cell Research | volume = 23 | issue = 10 | pages = 1229–1232 | date = October 2013 | pmid = 23958582 | pmc = 3790235 | doi = 10.1038/cr.2013.114 }}{{cite journal | vauthors = Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J | title = Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9 | journal = Nature Biotechnology | volume = 31 | issue = 8 | pages = 688–691 | date = August 2013 | pmid = 23929339 | pmc = 4078740 | doi = 10.1038/nbt.2654 }} It has since been successfully applied in several key crop species for the purpose of introducing or improving numerous agricultural traits.{{cite journal | vauthors = Zhu H, Li C, Gao C | title = Applications of CRISPR-Cas in agriculture and plant biotechnology | journal = Nature Reviews. Molecular Cell Biology | volume = 21 | issue = 11 | pages = 661–677 | date = November 2020 | pmid = 32973356 | doi = 10.1038/s41580-020-00288-9 | s2cid = 221918795 }} The development of CRISPR technology has been highly influential in the field of plant biotechnology, and has the potential to revolutionize the future of agriculture.

CRISPR-Cas systems are most commonly introduced into plants by Agrobacterium-mediated transformation, although particle bombardment and protoplast transformation are also used.{{cite journal | vauthors = Nadakuduti SS, Enciso-Rodríguez F | title = Advances in Genome Editing With CRISPR Systems and Transformation Technologies for Plant DNA Manipulation | language = English | journal = Frontiers in Plant Science | volume = 11 | pages = 637159 | date = 2021-01-14 | pmid = 33519884 | pmc = 7840963 | doi = 10.3389/fpls.2020.637159 | doi-access = free }}

Improvement of crop yields has been achieved in several species through the use of CRISPR-Cas technology. Grain yield in cereal crops is influenced by levels of the plant hormone cytokinin.{{cite journal | vauthors = Jameson PE, Song J | title = Will cytokinins underpin the second 'Green Revolution'? | journal = Journal of Experimental Botany | volume = 71 | issue = 22 | pages = 6872–6875 | date = December 2020 | pmid = 33382897 | doi = 10.1093/jxb/eraa447 | pmc = 8202814 }} High-yielding rice{{cite journal | vauthors = Zheng X, Zhang S, Liang Y, Zhang R, Liu L, Qin P, Zhang Z, Wang Y, Zhou J, Tang X, Zhang Y | title = Loss-function mutants of OsCKX gene family based on CRISPR-Cas systems revealed their diversified roles in rice | journal = The Plant Genome | volume = 16 | issue = 2 | pages = e20283 | date = June 2023 | pmid = 36660867 | doi = 10.1002/tpg2.20283 | doi-access = free }} and wheat{{cite journal | vauthors = Zhang Z, Hua L, Gupta A, Tricoli D, Edwards KJ, Yang B, Li W | title = Development of an Agrobacterium-delivered CRISPR/Cas9 system for wheat genome editing | journal = Plant Biotechnology Journal | volume = 17 | issue = 8 | pages = 1623–1635 | date = August 2019 | pmid = 30706614 | pmc = 6662106 | doi = 10.1111/pbi.13088 }} varieties have been produced by using CRISPR-Cas9 to knock out the enzyme cytokinin oxidase/dehydrogenase (CKX), which degrades cytokinin. Grain yield has also been increased in rice by using CRISPR-Cas9 to knock out an amino acid transporter.{{cite journal | vauthors = Lu K, Wu B, Wang J, Zhu W, Nie H, Qian J, Huang W, Fang Z | title = Blocking amino acid transporter OsAAP3 improves grain yield by promoting outgrowth buds and increasing tiller number in rice | journal = Plant Biotechnology Journal | volume = 16 | issue = 10 | pages = 1710–1722 | date = October 2018 | pmid = 29479779 | pmc = 6131477 | doi = 10.1111/pbi.12907 | s2cid = 3506253 | doi-access = free }}

CRISPR has been used to develop higher quality crops, including improvements to physical appearance, flavor and aroma, texture, shelf life, and nutritional content.{{cite journal | vauthors = Liu Q, Yang F, Zhang J, Liu H, Rahman S, Islam S, Ma W, She M | title = Application of CRISPR/Cas9 in Crop Quality Improvement | journal = International Journal of Molecular Sciences | volume = 22 | issue = 8 | pages = 4206 | date = April 2021 | pmid = 33921600 | doi = 10.3390/ijms22084206 | doi-access = free | pmc = 8073294 }} Pink,{{cite journal | vauthors = Yang T, Deng L, Zhao W, Zhang R, Jiang H, Ye Z, Li CB, Li C | title = Rapid breeding of pink-fruited tomato hybrids using the CRISPR/Cas9 system | journal = Journal of Genetics and Genomics = Yi Chuan Xue Bao | volume = 46 | issue = 10 | pages = 505–508 | date = October 2019 | pmid = 31734133 | doi = 10.1016/j.jgg.2019.10.002 }} yellow,{{cite journal | vauthors = Filler Hayut S, Melamed Bessudo C, Levy AA | title = Targeted recombination between homologous chromosomes for precise breeding in tomato | journal = Nature Communications | volume = 8 | issue = 1 | pages = 15605 | date = May 2017 | pmid = 28548094 | doi = 10.1038/ncomms15605 | pmc = 5458649 | bibcode = 2017NatCo...815605F }} and purple{{cite journal | vauthors = Vu TV, Sivankalyani V, Kim EJ, Doan DT, Tran MT, Kim J, Sung YW, Park M, Kang YJ, Kim JY | title = Highly efficient homology-directed repair using CRISPR/Cpf1-geminiviral replicon in tomato | journal = Plant Biotechnology Journal | volume = 18 | issue = 10 | pages = 2133–2143 | date = October 2020 | pmid = 32176419 | pmc = 7540044 | doi = 10.1111/pbi.13373 }} tomatoes have been produced by using CRISPR to mutate genes involved in synthesizing pigments. CRISPR has also been used to decrease starch content in wheat, thus improving grain quality.{{cite journal | vauthors = Zhang S, Zhang R, Gao J, Song G, Li J, Li W, Qi Y, Li Y, Li G | title = CRISPR/Cas9-mediated genome editing for wheat grain quality improvement | journal = Plant Biotechnology Journal | volume = 19 | issue = 9 | pages = 1684–1686 | date = September 2021 | pmid = 34143557 | pmc = 8428824 | doi = 10.1111/pbi.13647 }} In addition, soybeans have been modified using CRISPR to contain more heart-healthy monounsaturated fatty acids, like oleic acid.{{cite journal | vauthors = Do PT, Nguyen CX, Bui HT, Tran LT, Stacey G, Gillman JD, Zhang ZJ, Stacey MG | title = Demonstration of highly efficient dual gRNA CRISPR/Cas9 editing of the homeologous GmFAD2-1A and GmFAD2-1B genes to yield a high oleic, low linoleic and α-linolenic acid phenotype in soybean | journal = BMC Plant Biology | volume = 19 | issue = 1 | pages = 311 | date = July 2019 | pmid = 31307375 | pmc = 6632005 | doi = 10.1186/s12870-019-1906-8 | doi-access = free | bibcode = 2019BMCPB..19..311D }}

CRISPR technology has also been used to reduce the amount of allergens in foods.{{Cite journal | vauthors = Chakraborty A, Wylie SJ |date= December 2024 |title=Gene editing for allergen amelioration in plants – A review |journal=Plant Gene |volume=40 |pages=100476 |doi=10.1016/j.plgene.2024.100476 |bibcode=2024PlGen..4000476C |issn=2352-4073}} Wheat containing decreased levels of gluten, a common allergen and intolerance, has been developed using CRISPR.{{cite journal | vauthors = Sánchez-León S, Gil-Humanes J, Ozuna CV, Giménez MJ, Sousa C, Voytas DF, Barro F | title = Low-gluten, nontransgenic wheat engineered with CRISPR/Cas9 | journal = Plant Biotechnology Journal | volume = 16 | issue = 4 | pages = 902–910 | date = April 2018 | pmid = 28921815 | pmc = 5867031 | doi = 10.1111/pbi.12837 | s2cid = 4376988 | doi-access = free }} Researchers are also working to reduce allergens in soybean, peanut, and mustard using CRISPR-Cas9.

CRISPR has been used to develop plants with improved resistance to various diseases.{{cite journal | vauthors = Borrelli VM, Brambilla V, Rogowsky P, Marocco A, Lanubile A | title = The Enhancement of Plant Disease Resistance Using CRISPR/Cas9 Technology | language = English | journal = Frontiers in Plant Science | volume = 9 | pages = 1245 | date = 2018-08-24 | pmid = 30197654 | pmc = 6117396 | doi = 10.3389/fpls.2018.01245 | doi-access = free }} Using CRISPR, cucumber, rice, and tobacco plants have been engineered with resistance to viruses. Wheat, rice, tomato, grape, and cacao have been modified for resistance to fungal diseases. Finally, rice, apple, and citrus fruits have been developed with resistance to bacterial infection.

There are currently about 6000 known genetic disorders, most of which are currently untreatable. The role of CRISPR in gene therapy is to substitute exogenous DNA in place of defective genes.{{cite book | vauthors = Demirci S, Leonard A, Haro-Mora JJ, Uchida N, Tisdale JF | title = Cell Biology and Translational Medicine, Volume 5 | chapter = CRISPR/Cas9 for Sickle Cell Disease: Applications, Future Possibilities, and Challenges | series = Advances in Experimental Medicine and Biology | volume = 1144 | pages = 37–52 | date = 2019 | pmid = 30715679 | doi = 10.1007/5584_2018_331 | publisher = Springer International Publishing | isbn = 978-3-030-17588-7 | s2cid = 73432066 }} Gene therapy has made a huge impact and opened many new possibilities in medical biotechnology.{{cn|date=April 2025}}

There are two types of base editings:

Cytidine base editor is a novel therapy in which the cytidine (C) changes to thymidine (T).{{cn|date=April 2025}}

Adenine base editor (ABE),{{cite journal | vauthors = Fortunato F, Rossi R, Falzarano MS, Ferlini A | title = Innovative Therapeutic Approaches for Duchenne Muscular Dystrophy | journal = Journal of Clinical Medicine | volume = 10 | issue = 4 | page = 820 | date = February 2021 | pmid = 33671409 | pmc = 7922390 | doi = 10.3390/jcm10040820 | doi-access = free }} in this there is a change in base complements from adenine (A) to Guanine (G).

The mutations were directly installed in cellular DNA so that the donor template is not required. The base editings can only edit point mutations. Moreover, they can only fix up to four-point mutations.{{cite journal | vauthors = Stadtmauer EA, Fraietta JA, Davis MM, Cohen AD, Weber KL, Lancaster E, Mangan PA, Kulikovskaya I, Gupta M, Chen F, Tian L, Gonzalez VE, Xu J, Jung IY, Melenhorst JJ, Plesa G, Shea J, Matlawski T, Cervini A, Gaymon AL, Desjardins S, Lamontagne A, Salas-Mckee J, Fesnak A, Siegel DL, Levine BL, Jadlowsky JK, Young RM, Chew A, Hwang WT, Hexner EO, Carreno BM, Nobles CL, Bushman FD, Parker KR, Qi Y, Satpathy AT, Chang HY, Zhao Y, Lacey SF, June CH | title = CRISPR-engineered T cells in patients with refractory cancer | journal = Science | volume = 367 | issue = 6481 | date = February 2020 | pmid = 32029687 | pmc = 11249135 | doi = 10.1126/science.aba7365 | s2cid = 211048335 | doi-access = free }} To address this problem, the CRISPR system introduced a new technique known as Cas9 fusion to increase the scale of genes that can be edited.

Furthermore, the CRISPR Cas9 protein can modulate genes either by activating or silencing based on genes of interest.{{cite journal | vauthors = Jiang F, Doudna JA | title = CRISPR-Cas9 Structures and Mechanisms | journal = Annual Review of Biophysics | volume = 46 | issue = 1 | pages = 505–529 | date = May 2017 | pmid = 28375731 | doi = 10.1146/annurev-biophys-062215-010822 | s2cid = 274633 | doi-access = free }} There is a nuclease called dCas9 (endonuclease) used to silence or activate the expression of genes.{{cn|date=April 2025}}

The researchers are facing many challenges in gene editing.{{cite journal | vauthors = Zhang S, Shen J, Li D, Cheng Y | title = Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing | journal = Theranostics | volume = 11 | issue = 2 | pages = 614–648 | date = 2021 | pmid = 33391496 | pmc = 7738854 | doi = 10.7150/thno.47007 | s2cid = 226215184 | doi-access = free }} The major hurdles coming in the clinical applications are ethical issues and the transport system to the target site. As the units of CRISPR system taken from bacteria, when they are transferred to host cells it produces an immune response against them. Physical, chemical, viral vectors are used as vehicles to deliver the complex into the host.{{cn|date=January 2024}}

Due to this many complications are arising such as cell damage that leads to cell death. In the case of viral vectors, the capacity of the virus is small and Cas9 protein is large. So, to overcome these new methods were developed in which smaller strains of Cas9 are taken from bacteria. Finally, a great extent of work is still needed to improve the system.{{cn|date=April 2025}}

File:CRISPR diagnostics diagram.svg

File:F2. CRISPR.jpg

CRISPR associated nucleases have shown to be useful as a tool for molecular testing due to their ability to specifically target nucleic acid sequences in a high background of non-target sequences.{{cite journal | vauthors = Reis AC, Halper SM, Vezeau GE, Cetnar DP, Hossain A, Clauer PR, Salis HM | title = Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays | journal = Nature Biotechnology | volume = 37 | issue = 11 | pages = 1294–1301 | date = November 2019 | pmid = 31591552 | doi = 10.1038/s41587-019-0286-9 | osti = 1569832 | s2cid = 203852115 | url = https://www.osti.gov/biblio/1569832 | access-date = 2023-08-13 | archive-date = 2022-09-20 | archive-url = https://web.archive.org/web/20220920171246/https://www.osti.gov/biblio/1569832 | url-status = live }} In 2016, the Cas9 nuclease was used to deplete unwanted nucleotide sequences in next-generation sequencing libraries while requiring only 250 picograms of initial RNA input.{{cite journal | vauthors = Gu W, Crawford ED, O'Donovan BD, Wilson MR, Chow ED, Retallack H, DeRisi JL | title = Depletion of Abundant Sequences by Hybridization (DASH): using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications | journal = Genome Biology | volume = 17 | issue = 1 | page = 41 | date = March 2016 | pmid = 26944702 | pmc = 4778327 | doi = 10.1186/s13059-016-0904-5 | doi-access = free }} Beginning in 2017, CRISPR associated nucleases were also used for direct diagnostic testing of nucleic acids, down to single molecule sensitivity.{{cite journal | vauthors = Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F | title = Nucleic acid detection with CRISPR-Cas13a/C2c2 | journal = Science | volume = 356 | issue = 6336 | pages = 438–442 | date = April 2017 | pmid = 28408723 | pmc = 5526198 | doi = 10.1126/science.aam9321 | bibcode = 2017Sci...356..438G }}{{cite journal | vauthors = Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA | title = CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity | journal = Science | volume = 360 | issue = 6387 | pages = 436–439 | date = April 2018 | pmid = 29449511 | pmc = 6628903 | doi = 10.1126/science.aar6245 | bibcode = 2018Sci...360..436C }} CRISPR diversity is used as an analysis target to discern phylogeny and diversity in bacteria, such as in xanthomonads by Martins et al., 2019.{{cite book | vauthors = Shami A, Mostafa M, Abd-Elsalam KA | chapter = CRISPR Applications in Plant Bacteriology: today and future perspectives | veditors = Abd-Elsalam KA, Lim KT | title=CRISPR and RNAi Systems: Nanobiotechnology Approaches to Plant Breeding and Protection |location=Amsterdam | date=2021 | isbn=978-0-12-821911-9 | oclc=1240283203 | publisher=Elsevier | pages=xxxvi+804}}{{rp|page=552}} Early detections of plant pathogens by molecular typing of the pathogen's CRISPRs can be used in agriculture as demonstrated by Shen et al., 2020.{{rp|page=553}}

By coupling CRISPR-based diagnostics to additional enzymatic processes, the detection of molecules beyond nucleic acids is possible. One example of a coupled technology is SHERLOCK-based Profiling of IN vitro Transcription (SPRINT). SPRINT can be used to detect a variety of substances, such as metabolites in patient samples or contaminants in environmental samples, with high throughput or with portable point-of-care devices.{{cite journal | vauthors = Iwasaki RS, Batey RT | title = SPRINT: a Cas13a-based platform for detection of small molecules | journal = Nucleic Acids Research | volume = 48 | issue = 17 | pages = e101 | date = September 2020 | pmid = 32797156 | pmc = 7515716 | doi = 10.1093/nar/gkaa673 | doi-access = free }} CRISPR-Cas platforms are also being explored for detection{{cite journal | vauthors = Broughton JP, Deng X, Yu G, Fasching CL, Servellita V, Singh J, Miao X, Streithorst JA, Granados A, Sotomayor-Gonzalez A, Zorn K, Gopez A, Hsu E, Gu W, Miller S, Pan CY, Guevara H, Wadford DA, Chen JS, Chiu CY | title = CRISPR-Cas12-based detection of SARS-CoV-2 | journal = Nature Biotechnology | volume = 38 | issue = 7 | pages = 870–874 | date = July 2020 | pmid = 32300245 | doi = 10.1038/s41587-020-0513-4 | pmc = 9107629 | doi-access = free }}{{cite journal | vauthors = Nguyen LT, Smith BM, Jain PK | title = Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection | journal = Nature Communications | volume = 11 | issue = 1 | page = 4906 | date = September 2020 | pmid = 32999292 | doi = 10.1038/s41467-020-18615-1 | pmc = 7528031 | bibcode = 2020NatCo..11.4906N | doi-access = free }}{{cite journal | vauthors = Joung J, Ladha A, Saito M, Kim NG, Woolley AE, Segel M, Barretto RP, Ranu A, Macrae RK, Faure G, Ioannidi EI, Krajeski RN, Bruneau R, Huang MW, Yu XG, Li JZ, Walker BD, Hung DT, Greninger AL, Jerome KR, Gootenberg JS, Abudayyeh OO, Zhang F | title = Detection of SARS-CoV-2 with SHERLOCK One-Pot Testing | journal = The New England Journal of Medicine | volume = 383 | issue = 15 | pages = 1492–1494 | date = October 2020 | pmid = 32937062 | pmc = 7510942 | doi = 10.1056/NEJMc2026172 }}{{cite journal | vauthors = Patchsung M, Jantarug K, Pattama A, Aphicho K, Suraritdechachai S, Meesawat P, Sappakhaw K, Leelahakorn N, Ruenkam T, Wongsatit T, Athipanyasilp N, Eiamthong B, Lakkanasirorat B, Phoodokmai T, Niljianskul N, Pakotiprapha D, Chanarat S, Homchan A, Tinikul R, Kamutira P, Phiwkaow K, Soithongcharoen S, Kantiwiriyawanitch C, Pongsupasa V, Trisrivirat D, Jaroensuk J, Wongnate T, Maenpuen S, Chaiyen P, Kamnerdnakta S, Swangsri J, Chuthapisith S, Sirivatanauksorn Y, Chaimayo C, Sutthent R, Kantakamalakul W, Joung J, Ladha A, Jin X, Gootenberg JS, Abudayyeh OO, Zhang F, Horthongkham N, Uttamapinant C | title = Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA | journal = Nature Biomedical Engineering | volume = 4 | issue = 12 | pages = 1140–1149 | date = December 2020 | pmid = 32848209 | doi = 10.1038/s41551-020-00603-x | doi-access = free | hdl = 1721.1/138450.2 | hdl-access = free }} and inactivation of SARS-CoV-2, the virus that causes COVID-19.{{cite journal | vauthors = Konwarh R | title = Can CRISPR/Cas Technology Be a Felicitous Stratagem Against the COVID-19 Fiasco? Prospects and Hitches | journal = Frontiers in Molecular Biosciences | volume = 7 | page = 557377 | date = September 2020 | pmid = 33134311 | pmc = 7511716 | doi = 10.3389/fmolb.2020.557377 | doi-access = free }} Two different comprehensive diagnostic tests, AIOD-CRISPR and SHERLOCK test have been identified for SARS-CoV-2.{{cite journal | vauthors = Shademan B, Nourazarian A, Hajazimian S, Isazadeh A, Biray Avci C, Oskouee MA | title = CRISPR Technology in Gene-Editing-Based Detection and Treatment of SARS-CoV-2 | journal = Frontiers in Molecular Biosciences | volume = 8 | page = 772788 | date = 2022-01-11 | pmid = 35087864 | pmc = 8787289 | doi = 10.3389/fmolb.2021.772788 | doi-access = free }} The SHERLOCK test is based on a fluorescently labelled press reporter RNA which has the ability to identify 10 copies per microliter.{{cite journal | vauthors = Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F | title = SHERLOCK: nucleic acid detection with CRISPR nucleases | journal = Nature Protocols | volume = 14 | issue = 10 | pages = 2986–3012 | date = October 2019 | pmid = 31548639 | pmc = 6956564 | doi = 10.1038/s41596-019-0210-2 }} The AIOD-CRISPR helps with robust and highly sensitive visual detection of the viral nucleic acid.{{cite journal | vauthors = Ding X, Yin K, Li Z, Liu C | title = All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus | journal = bioRxiv | date = March 2020 | pmid = 32511323 | pmc = 7239053 | doi = 10.1101/2020.03.19.998724 }}

{{Further|Evolution of the brain#Genetic factors of recent evolution}}

CRISPR-Cas9 can be used in investigating and identifying the genetic differences of humans to other apes, especially of the brain. For example, by reintroducing archaic gene variants into brain organoids to show an impact on neurogenesis,{{cite journal | vauthors = Pinson A, Xing L, Namba T, Kalebic N, Peters J, Oegema CE, Traikov S, Reppe K, Riesenberg S, Maricic T, Derihaci R, Wimberger P, Pääbo S, Huttner WB | title = Human TKTL1 implies greater neurogenesis in frontal neocortex of modern humans than Neanderthals | journal = Science | volume = 377 | issue = 6611 | pages = eabl6422 | date = September 2022 | pmid = 36074851 | doi = 10.1126/science.abl6422 | s2cid = 252161562 }} metaphase length of apical progenitors of the developing neocortex,{{cite journal | vauthors = Mora-Bermúdez F, Kanis P, Macak D, Peters J, Naumann R, Xing L, Sarov M, Winkler S, Oegema CE, Haffner C, Wimberger P, Riesenberg S, Maricic T, Huttner WB, Pääbo S | title = Longer metaphase and fewer chromosome segregation errors in modern human than Neanderthal brain development | journal = Science Advances | volume = 8 | issue = 30 | pages = eabn7702 | date = July 2022 | pmid = 35905187 | pmc = 9337762 | doi = 10.1126/sciadv.abn7702 | bibcode = 2022SciA....8N7702M }} or by knockout of a gene in embryonic stem cells to identify a genetic regulator that via early cell shape transition drives evolutionary expansion of the human forebrain.{{cite news |title=Scientists discover how humans develop larger brains than other apes |url=https://phys.org/news/2021-03-scientists-humans-larger-brains-apes.html |access-date=19 April 2021 |work=phys.org |language=en |archive-date=19 April 2021 |archive-url=https://web.archive.org/web/20210419161119/https://phys.org/news/2021-03-scientists-humans-larger-brains-apes.html |url-status=live }}{{cite journal | vauthors = Benito-Kwiecinski S, Giandomenico SL, Sutcliffe M, Riis ES, Freire-Pritchett P, Kelava I, Wunderlich S, Martin U, Wray GA, McDole K, Lancaster MA | title = An early cell shape transition drives evolutionary expansion of the human forebrain | language = English | journal = Cell | volume = 184 | issue = 8 | pages = 2084–2102.e19 | date = April 2021 | pmid = 33765444 | pmc = 8054913 | doi = 10.1016/j.cell.2021.02.050 | doi-access = free }} 50px Available under [https://creativecommons.org/licenses/by/4.0/ CC BY 4.0] {{Webarchive|url=https://web.archive.org/web/20171016050101/https://creativecommons.org/licenses/by/4.0/ |date=2017-10-16 }}. One study described a major impact of an archaic gene variant on neurodevelopment{{cite news |title=Mini brains genetically altered with CRISPR to be Neanderthal-like |work=New Scientist |url=https://www.newscientist.com/article/2267610-mini-brains-genetically-altered-with-crispr-to-be-neanderthal-like/ |url-status=live |access-date=7 March 2021 |archive-url=https://web.archive.org/web/20210310060103/https://www.newscientist.com/article/2267610-mini-brains-genetically-altered-with-crispr-to-be-neanderthal-like/ |archive-date=10 March 2021 |vauthors=Sawal I}}{{cite journal | vauthors = Trujillo CA, Rice ES, Schaefer NK, Chaim IA, Wheeler EC, Madrigal AA, Buchanan J, Preissl S, Wang A, Negraes PD, Szeto RA, Herai RH, Huseynov A, Ferraz MS, Borges FS, Kihara AH, Byrne A, Marin M, Vollmers C, Brooks AN, Lautz JD, Semendeferi K, Shapiro B, Yeo GW, Smith SE, Green RE, Muotri AR | title = Reintroduction of the archaic variant of NOVA1 in cortical organoids alters neurodevelopment | journal = Science | volume = 371 | issue = 6530 | date = February 2021 | pmid = 33574182 | pmc = 8006534 | doi = 10.1126/science.aax2537 }} which may be an artefact of a CRISPR side effect,{{cite journal | vauthors = Maricic T, Helmbrecht N, Riesenberg S, Macak D, Kanis P, Lackner M, Pugach-Matveeva AD, Pääbo S | title = Comment on "Reintroduction of the archaic variant of NOVA1 in cortical organoids alters neurodevelopment" | journal = Science | volume = 374 | issue = 6565 | pages = eabi6060 | date = October 2021 | pmid = 34648345 | doi = 10.1126/science.abi6060 | s2cid = 238990790 }}{{cite journal | vauthors = Herai RH, Szeto RA, Trujillo CA, Muotri AR | title = Response to Comment on "Reintroduction of the archaic variant of NOVA1 in cortical organoids alters neurodevelopment" | journal = Science | volume = 374 | issue = 6565 | pages = eabi9881 | date = October 2021 | pmid = 34648331 | doi = 10.1126/science.abi9881 | s2cid = 238990560 }} as it could not be replicated in a subsequent study.

{{main|CRISPR interference}}

File:Dead-Cas9 potential applications.png

Using "dead" versions of Cas9 (dCas9) eliminates CRISPR's DNA-cutting ability, while preserving its ability to target desirable sequences. Multiple groups added various regulatory factors to dCas9s, enabling them to turn almost any gene on or off or adjust its level of activity.{{cite web | title = And Science's Breakthrough of the Year is ...|url = http://news.sciencemag.org/scientific-community/2015/12/and-science-s-breakthrough-year|website = news.sciencemag.org|access-date = 2015-12-21|date = December 17, 2015|last = Science News Staff}} Like RNAi, CRISPR interference (CRISPRi) turns off genes in a reversible fashion by targeting, but not cutting a site. The targeted site is methylated, epigenetically modifying the gene. This modification inhibits transcription. These precisely placed modifications may then be used to regulate the effects on gene expressions and DNA dynamics after the inhibition of certain genome sequences within DNA. Within the past few years, epigenetic marks in different human cells have been closely researched and certain patterns within the marks have been found to correlate with everything ranging from tumor growth to brain activity. Conversely, CRISPR-mediated activation (CRISPRa) promotes gene transcription.{{cite journal | vauthors = Dominguez AA, Lim WA, Qi LS | title = Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation | journal = Nature Reviews. Molecular Cell Biology | volume = 17 | issue = 1 | pages = 5–15 | date = January 2016 | pmid = 26670017 | pmc = 4922510 | doi = 10.1038/nrm.2015.2 }} Cas9 is an effective way of targeting and silencing specific genes at the DNA level.{{cite journal | vauthors = Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F | title = Genome-scale CRISPR-Cas9 knockout screening in human cells | journal = Science | volume = 343 | issue = 6166 | pages = 84–87 | date = January 2014 | pmid = 24336571 | pmc = 4089965 | doi = 10.1126/science.1247005 | bibcode = 2014Sci...343...84S }} In bacteria, the presence of Cas9 alone is enough to block transcription. For mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA sequences called promoters that immediately precede the target gene.{{cite journal | vauthors = Pennisi E | title = The CRISPR craze | journal = Science | volume = 341 | issue = 6148 | pages = 833–836 | date = August 2013 | pmid = 23970676 | doi = 10.1126/science.341.6148.833 | author-link = Elizabeth Pennisi | department = News Focus | bibcode = 2013Sci...341..833P }}

Cas9 was used to carry synthetic transcription factors that activated specific human genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different locations on the gene's promoter.

{{Main|RNA editing}}

In 2016, researchers demonstrated that CRISPR from an ordinary mouth bacterium could be used to edit RNA. The researchers searched databases containing hundreds of millions of genetic sequences for those that resembled CRISPR genes. They considered the fusobacterium Leptotrichia shahii. It had a group of genes that resembled CRISPR genes, but with important differences. When the researchers equipped other bacteria with these genes, which they called C2c2, they found that the organisms gained a novel defense.{{cite news | url = https://www.nytimes.com/2016/06/04/science/rna-c2c2-gene-editing-dna-crispr.html | title = Scientists Find Form of CRISPR Gene Editing With New Capabilities| vauthors = Zimmer C | date=2016-06-03 |newspaper=The New York Times |issn=0362-4331|access-date=2016-06-10}} C2c2 has later been renamed to Cas13a to fit the standard nomenclature for Cas genes.{{cite journal | vauthors = Pickar-Oliver A, Gersbach CA | title = The next generation of CRISPR-Cas technologies and applications | journal = Nature Reviews. Molecular Cell Biology | volume = 20 | issue = 8 | pages = 490–507 | date = August 2019 | pmid = 31147612 | pmc = 7079207 | doi = 10.1038/s41580-019-0131-5 }}

Many viruses encode their genetic information in RNA rather than DNA that they repurpose to make new viruses. HIV and poliovirus are such viruses. Bacteria with Cas13 make molecules that can dismember RNA, destroying the virus. Tailoring these genes opened any RNA molecule to editing.

CRISPR-Cas systems can also be employed for editing of micro-RNA and long-noncoding RNA genes in plants.{{cite journal | vauthors = Basak J, Nithin C | title = Targeting Non-Coding RNAs in Plants with the CRISPR-Cas Technology is a Challenge yet Worth Accepting | journal = Frontiers in Plant Science | volume = 6 | pages = 1001 | date = 2015 | pmid = 26635829 | pmc = 4652605 | doi = 10.3389/fpls.2015.01001 | doi-access = free }}

{{Excerpt|RNA editing|Therapeutic mRNA Editing}}

{{Excerpt|RNA editing|Comparison to DNA editing}}

{{Main|Gene drive}}

Gene drives may provide a powerful tool to restore balance of ecosystems by eliminating invasive species. Concerns regarding efficacy, unintended consequences in the target species as well as non-target species have been raised particularly in the potential for accidental release from laboratories into the wild. Scientists have proposed several safeguards for ensuring the containment of experimental gene drives including molecular, reproductive, and ecological.{{cite journal | vauthors = Akbari OS, Bellen HJ, Bier E, Bullock SL, Burt A, Church GM, Cook KR, Duchek P, Edwards OR, Esvelt KM, Gantz VM, Golic KG, Gratz SJ, Harrison MM, Hayes KR, James AA, Kaufman TC, Knoblich J, Malik HS, Matthews KA, O'Connor-Giles KM, Parks AL, Perrimon N, Port F, Russell S, Ueda R, Wildonger J | title = BIOSAFETY. Safeguarding gene drive experiments in the laboratory | journal = Science | volume = 349 | issue = 6251 | pages = 927–929 | date = August 2015 | pmid = 26229113 | pmc = 4692367 | doi = 10.1126/science.aac7932 | bibcode = 2015Sci...349..927A }} Many recommend that immunization and reversal drives be developed in tandem with gene drives in order to overwrite their effects if necessary.{{cite journal | vauthors = Caplan AL, Parent B, Shen M, Plunkett C | title = No time to waste—the ethical challenges created by CRISPR: CRISPR/Cas, being an efficient, simple, and cheap technology to edit the genome of any organism, raises many ethical and regulatory issues beyond the use to manipulate human germ line cells | journal = EMBO Reports | volume = 16 | issue = 11 | pages = 1421–1426 | date = November 2015 | pmid = 26450575 | pmc = 4641494 | doi = 10.15252/embr.201541337 }} There remains consensus that long-term effects must be studied more thoroughly particularly in the potential for ecological disruption that cannot be corrected with reversal drives.{{cite journal | vauthors = Oye KA, Esvelt K, Appleton E, Catteruccia F, Church G, Kuiken T, Lightfoot SB, McNamara J, Smidler A, Collins JP | title = Biotechnology. Regulating gene drives | journal = Science | volume = 345 | issue = 6197 | pages = 626–628 | date = August 2014 | pmid = 25035410 | doi = 10.1126/science.1254287 | doi-access = free | bibcode = 2014Sci...345..626O }}

Unenriched sequencing libraries often have abundant undesired sequences. Cas9 can specifically deplete the undesired sequences with double strand breakage with up to 99% efficiency and without significant off-target effects as seen with restriction enzymes. Treatment with Cas9 can deplete abundant rRNA while increasing pathogen sensitivity in RNA-seq libraries.{{cite journal | vauthors = Gu W, Crawford ED, O'Donovan BD, Wilson MR, Chow ED, Retallack H, DeRisi JL | title = Depletion of Abundant Sequences by Hybridization (DASH): using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications | journal = Genome Biology | volume = 17 | pages = 41 | date = March 2016 | pmid = 26944702 | pmc = 4778327 | doi = 10.1186/s13059-016-0904-5 | doi-access = free }}

{{Excerpt|Epigenome editing}}

{{Excerpt|Epigenome editing#Functional engineering}}

Combination of CRISPR-Cas9 with integrases enabled a technique for {{tooltip|large edits|"allowing large, multiplexed gene insertion without reliance on DNA repair pathways"}} without problematic double-stranded breaks, as demonstrated with {{tooltip|PASTE|Programmable Addition via Site-specific Targeting Elements}} in 2022. The researchers reported it could be used to deliver genes as long as 36,000 DNA base pairs to several types of human cells and thereby potentially for treating diseases caused by a large number of mutations.{{cite news | vauthors = McDonnell S |title=New CRISPR-based tool inserts large DNA sequences at desired sites in cells |url=https://phys.org/news/2022-11-crispr-based-tool-inserts-large-dna.html |access-date=18 December 2022 |work=Massachusetts Institute of Technology via phys.org |language=en}}{{cite journal | vauthors = Yarnall MT, Ioannidi EI, Schmitt-Ulms C, Krajeski RN, Lim J, Villiger L, Zhou W, Jiang K, Garushyants SK, Roberts N, Zhang L, Vakulskas CA, Walker JA, Kadina AP, Zepeda AE, Holden K, Ma H, Xie J, Gao G, Foquet L, Bial G, Donnelly SK, Miyata Y, Radiloff DR, Henderson JM, Ujita A, Abudayyeh OO, Gootenberg JS | title = Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases | journal = Nature Biotechnology | pages = 500–512 | date = November 2022 | volume = 41 | issue = 4 | pmid = 36424489 | doi = 10.1038/s41587-022-01527-4 | pmc = 10257351 | s2cid = 253879386 | biorxiv = 10.1101/2021.11.01.466786 }}

{{Main|Prime editing}}

Prime editing{{cite journal | vauthors = Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR | title = Search-and-replace genome editing without double-strand breaks or donor DNA | journal = Nature | volume = 576 | issue = 7785 | pages = 149–157 | date = December 2019 | pmid = 31634902 | pmc = 6907074 | doi = 10.1038/s41586-019-1711-4 | bibcode = 2019Natur.576..149A }} (or base editing) is a CRISPR refinement to accurately insert or delete sections of DNA. The CRISPR edits are not always perfect and the cuts can end up in the wrong place. Both issues are a problem for using the technology in medicine.{{cite web | url = https://www.smithsonianmag.com/science-nature/prime-editing-new-form-crispr-technology-make-gene-editing-more-precisie-180973381/ | title = A New Gene Editing Tool Could Make CRISPR More Precise. | vauthors = Thulin L | work = The Smithsonian Magazine | date = 21 October 2019 }} Prime editing does not cut the double-stranded DNA but instead uses the CRISPR targeting apparatus to shuttle an additional enzyme to a desired sequence, where it converts a single nucleotide into another.{{cite web | url = https://www.science.org/content/article/new-prime-genome-editor-could-surpass-crispr | title = New 'prime' genome editor could surpass CRISPR. | vauthors = Cohen J | work = Science Magazine | date = 21 October 2019 }} The new guide, called a pegRNA, contains an RNA template for a new DNA sequence to be added to the genome at the target location. That requires a second protein, attached to Cas9: a reverse transcriptase enzyme, which can make a new DNA strand from the RNA template and insert it at the nicked site.{{cite web | url = https://www.the-scientist.com/news-opinion/new-prime-editing-method-makes-only-single-stranded-dna-cuts-66608 | title = New "Prime Editing" Method Makes Only Single-Stranded DNA Cuts. | vauthors = Yasinski E | work = The Scientist | date = 21 October 2019 }} Those three independent pairing events each provide an opportunity to prevent off-target sequences, which significantly increases targeting flexibility and editing precision. Prime editing was developed by researchers at the Broad Institute of MIT and Harvard in Massachusetts. More work is needed to optimize the methods.{{cite web | url = https://www.bbc.com/news/health-50125843 | title = Prime editing: DNA tool could correct 89% of genetic defects | vauthors = Gallagher J | work = BBC News | date = 21 October 2019 }}

As of March 2015, multiple groups had announced ongoing research with the intention of laying the foundations for applying CRISPR to human embryos for human germline engineering, including labs in the US, China, and the UK, as well as US biotechnology company OvaScience.{{cite journal | vauthors = Regalado A | journal = MIT Technology Review | date = March 5, 2015 | url = http://www.technologyreview.com/featuredstory/535661/engineering-the-perfect-baby/ | title = Engineering the Perfect Baby |archive-url=https://web.archive.org/web/20150313092113/https://www.technologyreview.com/featuredstory/535661/engineering-the-perfect-baby/ |archive-date= March 13, 2015 |url-status=dead}} Scientists, including a CRISPR co-discoverer, urged a worldwide moratorium on applying CRISPR to the human germline, especially for clinical use. They said "scientists should avoid even attempting, in lax jurisdictions, germline genome modification for clinical application in humans" until the full implications "are discussed among scientific and governmental organizations".{{cite journal | vauthors = Baltimore D, Berg P, Botchan M, Carroll D, Charo RA, Church G, Corn JE, Daley GQ, Doudna JA, Fenner M, Greely HT, Jinek M, Martin GS, Penhoet E, Puck J, Sternberg SH, Weissman JS, Yamamoto KR | title = Biotechnology. A prudent path forward for genomic engineering and germline gene modification | journal = Science | volume = 348 | issue = 6230 | pages = 36–38 | date = April 2015 | pmid = 25791083 | pmc = 4394183 | doi = 10.1126/science.aab1028 | bibcode = 2015Sci...348...36B }}{{cite journal | vauthors = Lanphier E, Urnov F, Haecker SE, Werner M, Smolenski J | title = Don't edit the human germ line | journal = Nature | volume = 519 | issue = 7544 | pages = 410–411 | date = March 2015 | pmid = 25810189 | doi = 10.1038/519410a | doi-access = free | bibcode = 2015Natur.519..410L }} These scientists support further low-level research on CRISPR and do not see CRISPR as developed enough for any clinical use in making heritable changes to humans.{{cite news | vauthors = Wade N | title = Scientists Seek Ban on Method of Editing the Human Genome | url = https://www.nytimes.com/2015/03/20/science/biologists-call-for-halt-to-gene-editing-technique-in-humans.html | date =19 March 2015 | work = The New York Times | access-date = 20 March 2015 | quote = The biologists writing in Science support continuing laboratory research with the technique, and few if any scientists believe it is ready for clinical use.}}

In April 2015, Chinese scientists reported results of an attempt to alter the DNA of non-viable human embryos using CRISPR to correct a mutation that causes beta thalassemia, a lethal heritable disorder.{{cite journal | vauthors = Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, Lv J, Xie X, Chen Y, Li Y, Sun Y, Bai Y, Songyang Z, Ma W, Zhou C, Huang J | title = CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes | journal = Protein & Cell | volume = 6 | issue = 5 | pages = 363–372 | date = May 2015 | pmid = 25894090 | pmc = 4417674 | doi = 10.1007/s13238-015-0153-5 }}{{cite news | vauthors = Kolata G | title=Chinese Scientists Edit Genes of Human Embryos, Raising Concerns |url=https://www.nytimes.com/2015/04/24/health/chinese-scientists-edit-genes-of-human-embryos-raising-concerns.html |date=23 April 2015 |work=The New York Times |access-date=24 April 2015 }} The study had previously been rejected by both Nature and Science in part because of ethical concerns.{{cite journal |doi=10.1038/nature.2015.17378 |title=Chinese scientists genetically modify human embryos |journal=Nature |year=2015 | vauthors = Cyranoski D, Reardon S |s2cid=87604469 }} The experiments resulted in successfully changing only some of the intended genes, and had off-target effects on other genes. The researchers stated that CRISPR is not ready for clinical application in reproductive medicine. In April 2016, Chinese scientists were reported to have made a second unsuccessful attempt to alter the DNA of non-viable human embryos using CRISPR – this time to alter the CCR5 gene to make the embryo resistant to HIV infection.{{Cite web|url=https://www.technologyreview.com/s/601235/chinese-researchers-experiment-with-making-hiv-proof-embryos/|title=Chinese Researchers Experiment with Making HIV-Proof Embryos| vauthors = Regalado A |date=2016-05-08|website=MIT Technology Review|access-date=2016-06-10}}

In December 2015, an International Summit on Human Gene Editing took place in Washington under the guidance of David Baltimore. Members of national scientific academies of the US, UK, and China discussed the ethics of germline modification. They agreed to support basic and clinical research under certain legal and ethical guidelines. A specific distinction was made between somatic cells, where the effects of edits are limited to a single individual, and germline cells, where genome changes can be inherited by descendants. Heritable modifications could have unintended and far-reaching consequences for human evolution, genetically (e.g. gene–environment interactions) and culturally (e.g. social Darwinism). Altering of gametocytes and embryos to generate heritable changes in humans was defined to be irresponsible. The group agreed to initiate an international forum to address such concerns and harmonize regulations across countries.{{cite web | url = http://www8.nationalacademies.org/onpinews/newsitem.aspx?RecordID=12032015a | date =3 December 2015 | title = International Summit on Gene Editing | publisher =National Academies of Sciences, Engineering, and Medicine | access-date = 3 December 2015 }}

In February 2017, the United States National Academies of Sciences, Engineering, and Medicine (NASEM) Committee on Human Gene Editing published a report reviewing ethical, legal, and scientific concerns of genomic engineering technology. The conclusion of the report stated that heritable genome editing is impermissible now but could be justified for certain medical conditions; however, they did not justify the usage of CRISPR for enhancement.{{cite journal | vauthors = Brokowski C | title = Do CRISPR Germline Ethics Statements Cut It? | journal = The CRISPR Journal | volume = 1 | issue = 2 | pages = 115–125 | date = April 2018 | pmid = 31021208 | pmc = 6694771 | doi = 10.1089/crispr.2017.0024 }}

In November 2018, Jiankui He announced that he had edited two human embryos to attempt to disable the gene for CCR5, which codes for a receptor that HIV uses to enter cells. He said that twin girls, Lulu and Nana, had been born a few weeks earlier. He said that the girls still carried functional copies of CCR5 along with disabled CCR5 (mosaicism) and were still vulnerable to HIV. The work was widely condemned as unethical, dangerous, and premature.{{cite news | vauthors = Begley S |title=Amid uproar, Chinese scientist defends creating gene-edited babies |url=https://www.statnews.com/2018/11/28/chinese-scientist-defends-creating-gene-edited-babies/ |work=STAT |date=28 November 2018}} An international group of scientists called for a global moratorium on genetically editing human embryos.{{cite web|url=https://www.theguardian.com/science/2019/mar/13/scientists-call-for-global-moratorium-on-crispr-gene-editing|title=Scientists call for global moratorium on gene editing of embryos| vauthors = Sample I |date=13 March 2019|access-date=14 March 2019|website=Theguardian.com}}

Designer babies

The advent of CRISPR-Cas9 gene editing technology has led to the possibility of creating "designer babies." This technology has the possibility of eliminating certain genetic diseases, or improving health by enhancing certain genetic traits.{{cn|date=April 2025}}

Policy regulations for the CRISPR-Cas9 system vary around the globe. In February 2016, British scientists were given permission by regulators to genetically modify human embryos by using CRISPR-Cas9 and related techniques. However, researchers were forbidden from implanting the embryos and the embryos were to be destroyed after seven days.{{cite journal | vauthors = Callaway E | title = UK scientists gain licence to edit genes in human embryos | journal = Nature | volume = 530 | issue = 7588 | pages = 18 | date = February 2016 | pmid = 26842037 | doi = 10.1038/nature.2016.19270 | doi-access = free | bibcode = 2016Natur.530...18C }}

The US has an elaborate, interdepartmental regulatory system to evaluate new genetically modified foods and crops. For example, the Agriculture Risk Protection Act of 2000 gives the United States Department of Agriculture the authority to oversee the detection, control, eradication, suppression, prevention, or retardation of the spread of plant pests or noxious weeds to protect the agriculture, environment, and economy of the US. The act regulates any genetically modified organism that utilizes the genome of a predefined "plant pest" or any plant not previously categorized.{{cite journal | vauthors = McHughen A, Smyth S | title = US regulatory system for genetically modified [genetically modified organism (GMO), rDNA or transgenic] crop cultivars | journal = Plant Biotechnology Journal | volume = 6 | issue = 1 | pages = 2–12 | date = January 2008 | pmid = 17956539 | doi = 10.1111/j.1467-7652.2007.00300.x | s2cid = 3210837 | doi-access = free }} In 2015, Yinong Yang successfully deactivated 16 specific genes in the white button mushroom to make them non-browning. Since he had not added any foreign-species (transgenic) DNA to his organism, the mushroom could not be regulated by the USDA under Section 340.2.{{cite web|url=https://www.aphis.usda.gov/biotechnology/downloads/reg_loi/15-321-01_air_response_signed.pdf|title=Re: Request to confirm |author=USDA| author-link=USDA}} Yang's white button mushroom was the first organism genetically modified with the CRISPR-Cas9 protein system to pass US regulation.{{cite journal | vauthors = Waltz E | title = Gene-edited CRISPR mushroom escapes US regulation | journal = Nature | volume = 532 | issue = 7599 | pages = 293 | date = April 2016 | pmid = 27111611 | doi = 10.1038/nature.2016.19754 | doi-access = free | bibcode = 2016Natur.532..293W }}

In 2016, the USDA sponsored a committee to consider future regulatory policy for upcoming genetic modification techniques. With the help of the US National Academies of Sciences, Engineering, and Medicine, special interests groups met on April 15 to contemplate the possible advancements in genetic engineering within the next five years and any new regulations that might be needed as a result.{{cite journal | vauthors = Ledford H | title = Gene-editing surges as US rethinks regulations | journal = Nature | volume = 532 | issue = 7598 | pages = 158–159 | date = April 2016 | pmid = 27075074 | doi = 10.1038/532158a | doi-access = free | bibcode = 2016Natur.532..158L }} In 2017, the Food and Drug Administration proposed a rule that would classify genetic engineering modifications to animals as "animal drugs", subjecting them to strict regulation if offered for sale and reducing the ability for individuals and small businesses to make them profitable.{{cite web | url = http://www.gizmodo.com.au/2017/02/the-fda-is-cracking-down-on-rogue-genetic-engineers/ | title = The FDA Is Cracking Down On Rogue Genetic Engineers | vauthors = Brown KV | work = Gizmodo | date = 1 February 2017 | access-date = 5 February 2017 }}{{cite web |url= https://www.fda.gov/downloads/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/UCM113903.pdf |archive-url= https://web.archive.org/web/20090709170807/http://www.fda.gov/downloads/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/UCM113903.pdf |url-status= dead |archive-date= July 9, 2009 |title=Guidance for Industry #187 / Regulation of Intentionally Altered Genomic DNA in Animals |website=Food and Drug Administration |date=2020-02-11 }}

In China, where social conditions sharply contrast with those of the West, genetic diseases carry a heavy stigma.{{cite journal | vauthors = Cyranoski D | title = China's embrace of embryo selection raises thorny questions | journal = Nature | volume = 548 | issue = 7667 | pages = 272–274 | date = August 2017 | pmid = 28816265 | doi = 10.1038/548272a | doi-access = free | bibcode = 2017Natur.548..272C }} This leaves China with fewer policy barriers to the use of this technology.{{cite journal | vauthors = Peng Y | title = The morality and ethics governing CRISPR-Cas9 patents in China | journal = Nature Biotechnology | volume = 34 | issue = 6 | pages = 616–618 | date = June 2016 | pmid = 27281418 | doi = 10.1038/nbt.3590 | s2cid = 38509820 }}{{cite news | url = https://www.wsj.com/articles/china-unhampered-by-rules-races-ahead-in-gene-editing-trials-1516562360 | title = China, Unhampered by Rules, Races Ahead in Gene-Editing Trials | vauthors = Rana P, Marcus A, Fan W | date = 2018-01-21 | work = Wall Street Journal | access-date = 2018-01-23 | issn = 0099-9660 }}

In 2012 and 2013, CRISPR was a runner-up in Science Magazine's Breakthrough of the Year award. In 2015, it was the winner of that award. CRISPR was named as one of MIT Technology Review{{'}}s 10 breakthrough technologies in 2014 and 2016.{{cite news | vauthors = Talbot D | title = Precise Gene Editing in Plants/ 10 Breakthrough Technologies 2016 |url=https://www.technologyreview.com/s/600765/10-breakthrough-technologies-2016-precise-gene-editing-in-plants/ |access-date=18 March 2016

|archive-url=https://web.archive.org/web/20171105023813/https://www.technologyreview.com/s/600765/10-breakthrough-technologies-2016-precise-gene-editing-in-plants/ |archive-date=November 5, 2017 |work=MIT Technology review | publisher = Massachusetts Institute of Technology|date=2016}}{{cite news | vauthors = Larson C, Schaffer A | title=Genome Editing/ 10 Breakthrough Technologies 2014

|url=https://www.technologyreview.com/s/526511/genome-editing/ | access-date=18 March 2016

|archive-url=https://web.archive.org/web/20161205081333/https://www.technologyreview.com/s/526511/genome-editing/

|archive-date=December 5, 2016

|publisher=Massachusetts Institute of Technology | date = 2014 }} In 2016, Jennifer Doudna and Emmanuelle Charpentier, along with Rudolph Barrangou, Philippe Horvath, and Feng Zhang won the Gairdner International award. In 2017, Doudna and Charpentier were awarded the Japan Prize in Tokyo, Japan for their revolutionary invention of CRISPR-Cas9. In 2016, Charpentier, Doudna, and Zhang won the Tang Prize in Biopharmaceutical Science.{{Cite web|url=http://www.tang-prize.org/en/owner_detail.php?cat=11&id=554|title=Tang Prize Laureates|website=www.tang-prize.org|language=en|access-date=2018-08-05}} In 2020, Charpentier and Doudna were awarded the Nobel Prize in Chemistry, "for the development of a method for genome editing."{{cite web |title=Press release: The Nobel Prize in Chemistry 2020 |url=https://www.nobelprize.org/prizes/chemistry/2020/press-release/ |publisher=Nobel Foundation |access-date=7 October 2020}}

{{Portal|Biology|Technology}}

{{div col|colwidth=20em}}

• CRISPR/Cas Tools
• The CRISPR Journal
• Eugenics
• DRACO
• Zinc finger
• Gene knockout
• Genetics
• Glossary of genetics
• Human Nature (2019 documentary film)
• LEAPER gene editing
• Make People Better (2022 documentary)
• RNAi
• SiRNA
• Surveyor nuclease assay
• Synthetic biology

{{div col end}}

{{Reflist}}

{{Breakthrough of the Year}}

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Category:Biotechnology

Category:Genetic engineering

Category:Genome editing