cas9

The Cas9 endonuclease is a four-component system that includes two small molecules: crRNA and trans-activating CRISPR RNA (tracrRNA).{{cite journal | vauthors = Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E | title = CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III | journal = Nature | volume = 471 | issue = 7340 | pages = 602–607 | date = March 2011 | pmid = 21455174 | pmc = 3070239 | doi = 10.1038/nature09886 | bibcode = 2011Natur.471..602D }}{{cite journal | vauthors = Barrangou R | author-link=Rodolphe Barrangou | title = Diversity of CRISPR-Cas immune systems and molecular machines | journal = Genome Biology | volume = 16 | page = 247 | date = November 2015 | pmid = 26549499 | pmc = 4638107 | doi = 10.1186/s13059-015-0816-9 | doi-access=free }} In 2012, Jennifer Doudna and Emmanuelle Charpentier re-engineered the Cas9 endonuclease into a more manageable two-component system by fusing the two RNA molecules into a "single-guide RNA" that, when combined with Cas9, could find and cut the DNA target specified by the guide RNA.{{cite journal | vauthors = Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E | author-link5=Jennifer Doudna | title = A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity | journal = Science | volume = 337 | issue = 6096 | pages = 816–821 | date = August 2012 | pmid = 22745249 | pmc = 6286148| doi = 10.1126/science.1225829 | bibcode = 2012Sci...337..816J }} This contribution was so significant that it was recognized by the Nobel Prize in Chemistry in 2020. By manipulating the nucleotide sequence of the guide RNA, the artificial Cas9 system could be programmed to target any DNA sequence for separation. Another collaboration comprising Virginijus Šikšnys, Gasiūnas, Barrangou, and Horvath showed that Cas9 from the S. thermophilus CRISPR system can also be reprogrammed to target a site of their choosing by changing the sequence of its crRNA. These advances fueled efforts to edit genomes with the modified CRISPR-Cas9 system.{{cite journal |vauthors=Mojica FJ, Montoliu L |title=On the Origin of CRISPR-Cas Technology: From Prokaryotes to Mammals |journal=Trends in Microbiology |volume=24 |issue=10 |pages=811–820 |year=2016 |pmid=27401123 |doi=10.1016/j.tim.2016.06.005 }}

Groups led by Feng Zhang and George Church simultaneously published descriptions of genome editing in human cell cultures using CRISPR-Cas9 for the first time.{{cite journal | vauthors = Hsu PD, Lander ES, Zhang F | author-link3=Feng Zhang | title = Development and applications of CRISPR-Cas9 for genome engineering | journal = Cell | volume = 157 | issue = 6 | pages = 1262–1278 | date = June 2014 | pmid = 24906146 | pmc = 4343198 | doi = 10.1016/j.cell.2014.05.010 }}{{cite journal | vauthors = Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F | author-link11=Feng Zhang | title = Multiplex genome engineering using CRISPR/Cas systems | journal = Science | volume = 339 | issue = 6121 | pages = 819–823 | date = February 2013 | pmid = 23287718 | pmc = 3795411 | doi = 10.1126/science.1231143 | bibcode = 2013Sci...339..819C }}{{cite journal | vauthors = Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM | title = RNA-guided human genome engineering via Cas9 | journal = Science | volume = 339 | issue = 6121 | pages = 823–826 | date = February 2013 | pmid = 23287722 | pmc = 3712628 | doi = 10.1126/science.1232033 | bibcode = 2013Sci...339..823M }} It has since been used in a wide range of organisms, including baker's yeast (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 Research | volume = 41 | issue = 7 | pages = 4336–4343 | date = April 2013 | pmid = 23460208 | pmc = 3627607 | doi = 10.1093/nar/gkt135 }}{{cite journal | vauthors = Zhang GC, Kong II, Kim H, Liu JJ, Cate JH, Jin YS | title = Construction of a quadruple auxotrophic mutant of an industrial polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease | journal = Applied and Environmental Microbiology | volume = 80 | issue = 24 | pages = 7694–7701 | date = December 2014 | pmid = 25281382 | pmc = 4249234 | doi = 10.1128/AEM.02310-14 | bibcode = 2014ApEnM..80.7694Z }}{{cite journal | vauthors = Liu JJ, Kong II, Zhang GC, Jayakody LN, Kim H, Xia PF, Kwak S, Sung BH, Sohn JH, Walukiewicz HE, Rao CV, Jin YS | title = Metabolic Engineering of Probiotic Saccharomyces boulardii | journal = Applied and Environmental Microbiology | volume = 82 | issue = 8 | pages = 2280–2287 | date = April 2016 | pmid = 26850302 | pmc = 4959471 | doi = 10.1128/AEM.00057-16 | bibcode = 2016ApEnM..82.2280L }} the opportunistic pathogen Candida albicans,{{cite journal | vauthors = Vyas VK, Barrasa MI, Fink GR | title = Candida albicans CRISPR system permits genetic engineering of essential genes and gene families | journal = Science Advances | volume = 1 | issue = 3 | pages = e1500248 | date = 2015 | pmid = 25977940 | pmc = 4428347 | doi = 10.1126/sciadv.1500248 | bibcode = 2015SciA....1E0248V }}{{cite journal | vauthors = Ng H, Dean N | title = Candida albicans by Increased Single Guide RNA Expression | journal = mSphere | volume = 2 | issue = 2 | pages = e00385–16 | year = 2017 | pmid = 28435892 | pmc = 5397569 | doi = 10.1128/mSphere.00385-16 }} zebrafish (Danio rerio),{{cite journal | vauthors = Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK | title = Efficient genome editing in zebrafish using a CRISPR-Cas system | journal = Nature Biotechnology | volume = 31 | issue = 3 | pages = 227–229 | date = March 2013 | pmid = 23360964 | pmc = 3686313 | doi = 10.1038/nbt.2501 }} fruit flies (Drosophila melanogaster),{{cite journal | vauthors = Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O'Connor-Giles KM | title = Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease | journal = Genetics | volume = 194 | issue = 4 | pages = 1029–1035 | date = August 2013 | pmid = 23709638 | pmc = 3730909 | doi = 10.1534/genetics.113.152710 }}{{cite journal | vauthors = Bassett AR, Tibbit C, Ponting CP, Liu JL | title = Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system | journal = Cell Reports | volume = 4 | issue = 1 | pages = 220–228 | date = July 2013 | pmid = 23827738 | pmc = 3714591 | doi = 10.1016/j.celrep.2013.06.020 }} ants (Harpegnathos saltator{{cite journal | vauthors = Yan H, Opachaloemphan C, Mancini G, Yang H, Gallitto M, Mlejnek J, Leibholz A, Haight K, Ghaninia M, Huo L, Perry M, Slone J, Zhou X, Traficante M, Penick CA, Dolezal K, Gokhale K, Stevens K, Fetter-Pruneda I, Bonasio R, Zwiebel LJ, Berger SL, Liebig J, Reinberg D, Desplan C | title = An Engineered orco Mutation Produces Aberrant Social Behavior and Defective Neural Development in Ants | journal = Cell | volume = 170 | issue = 4 | pages = 736–747.e9 | date = August 2017 | pmid = 28802043 | pmc = 5587193 | doi = 10.1016/j.cell.2017.06.051 }} and Ooceraea biroi{{cite journal | vauthors = Trible W, Olivos-Cisneros L, McKenzie SK, Saragosti J, Chang NC, Matthews BJ, Oxley PR, Kronauer DJ | title = orco Mutagenesis Causes Loss of Antennal Lobe Glomeruli and Impaired Social Behavior in Ants | journal = Cell | volume = 170 | issue = 4 | pages = 727–735.e10 | date = August 2017 | pmid = 28802042 | pmc = 5556950 | doi = 10.1016/j.cell.2017.07.001 }}), mosquitoes (Aedes aegypti{{cite journal | vauthors = Kistler KE, Vosshall LB, Matthews BJ | title = Genome engineering with CRISPR-Cas9 in the mosquito Aedes aegypti | journal = Cell Reports | volume = 11 | issue = 1 | pages = 51–60 | date = April 2015 | pmid = 25818303 | pmc = 4394034 | doi = 10.1016/j.celrep.2015.03.009 }}), nematodes (Caenorhabditis elegans),{{cite journal | vauthors = Friedland AE, Tzur YB, Esvelt KM, Colaiácovo MP, Church GM, Calarco JA | title = Heritable genome editing in C. elegans via a CRISPR-Cas9 system | journal = Nature Methods | volume = 10 | issue = 8 | pages = 741–743 | date = August 2013 | pmid = 23817069 | pmc = 3822328 | doi = 10.1038/nmeth.2532 }} plants,{{cite journal | vauthors = Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP | title = Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice | journal = Nucleic Acids Research | volume = 41 | issue = 20 | pages = e188 | date = November 2013 | pmid = 23999092 | pmc = 3814374 | doi = 10.1093/nar/gkt780 }} mice (Mus musculus domesticus),{{cite journal | vauthors = Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R | author-link6=Feng Zhang | title = One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering | journal = Cell | volume = 153 | issue = 4 | pages = 910–918 | date = May 2013 | pmid = 23643243 | pmc = 3969854 | doi = 10.1016/j.cell.2013.04.025 }}{{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 | page = 60| date = May 2018 | pmid = 29796309| pmc =5955943 | doi = 10.1038/s41420-018-0056-3}} monkeys{{cite journal | vauthors = Guo X, Li XJ | title = Targeted genome editing in primate embryos | journal = Cell Research | volume = 25 | issue = 7 | pages = 767–768 | date = July 2015 | pmid = 26032266 | pmc = 4493275 | doi = 10.1038/cr.2015.64 }} and human embryos.{{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 | author-link9=Jennifer Doudna | 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 }}

CRISPR has been modified to make programmable transcription factors that allow activation or silencing of targeted genes.{{cite journal | vauthors = Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS | title = CRISPR interference (CRISPRi) for sequence-specific control of gene expression | journal = Nature Protocols | volume = 8 | issue = 11 | pages = 2180–2196 | date = November 2013|pmc=3922765| pmid = 24136345 | doi = 10.1038/nprot.2013.132 }}

File:Cas12a vs Cas9 cleavage position.svg and Cas9 with the position of DNA cleavage shown relative to their PAM sequences in a zoom-in]]

The CRISPR-Cas9 system has been shown to make effective gene edits in Human tripronuclear zygotes, as first described in a 2015 paper by Chinese scientists P. Liang and Y. Xu. The system made a successful cleavage of mutant Beta-Hemoglobin (HBB) in 28 out of 54 embryos. Four out of the 28 embryos were successfully recombined using a donor template. The scientists showed that during DNA recombination of the cleaved strand, the homologous endogenous sequence HBD competes with the exogenous donor template. DNA repair in human embryos is much more complicated and particular than in derived stem cells.{{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 | doi = 10.1007/s13238-015-0153-5 | pmid=25894090 | pmc=4417674}}

To survive in a variety of challenging, inhospitable habitats that are filled with bacteriophages, bacteria and archaea have evolved methods to evade and fend off predatory viruses. This includes the CRISPR system of adaptive immunity. In practice, CRISPR/Cas systems act as self-programmable restriction enzymes. CRISPR loci are composed of short, palindromic repeats that occur at regular intervals composed of alternate CRISPR repeats and variable CRISPR spacers between 24 and 48 nucleotides long. These CRISPR loci are usually accompanied by adjacent CRISPR-associated (cas) genes. In 2005, it was discovered by three separate groups that the spacer regions were homologous to foreign DNA elements, including plasmids and viruses. These reports provided the first biological evidence that CRISPRs might function as an immune system.

Cas9 has been used often as a genome-editing tool. Cas9 has been used in recent developments in preventing viruses from manipulating hosts' DNA. Since the CRISPR-Cas9 was developed from bacterial genome systems, it can be used to target the genetic material in viruses. The use of the enzyme Cas9 can be a solution to many viral infections. Cas9 possesses the ability to target specific viruses by the targeting of specific strands of the viral genetic information. More specifically the Cas9 enzyme targets certain sections of the viral genome that prevents the virus from carrying out its normal function.{{Cite book |title=CRISPR-Cas: a laboratory manual |isbn=9781621821304|location=Cold Spring Harbor, New York|oclc=922914104| vauthors = Doudna JA, Mali P |year=2016}} Cas9 has also been used to disrupt the detrimental strand of DNA and RNA that cause diseases and mutated strands of DNA. Cas9 has already showed promise in disrupting the effects of HIV-1. Cas9 has been shown to suppress the expression of the long terminal repeats in HIV-1. When introduced into the HIV-1 genome Cas9 has shown the ability to mutate strands of HIV-1.{{cite journal | vauthors = Chen W, Page-McCaw PS | date = March 2019 | title = CRISPR/Cas9 gene editing. | journal = AccessScience | publisher = McGraw-Hill Education | doi = 10.1036/1097-8542.168060 }}{{cite journal | vauthors = Ebina H, Misawa N, Kanemura Y, Koyanagi Y | title = Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus | journal = Scientific Reports | volume = 3 | issue = 1 | pages = 2510 | date = 2013-08-26 | pmid = 23974631 | pmc = 3752613 | doi = 10.1038/srep02510 | bibcode = 2013NatSR...3.2510E }} Cas9 has also been used in the treatment of Hepatitis B through targeting of the ends of certain of long terminal repeats in the Hepatitis B viral genome.{{cite journal | vauthors = Li H, Sheng C, Wang S, Yang L, Liang Y, Huang Y, Liu H, Li P, Yang C, Yang X, Jia L, Xie J, Wang L, Hao R, Du X, Xu D, Zhou J, Li M, Sun Y, Tong Y, Li Q, Qiu S, Song H | title = Removal of Integrated Hepatitis B Virus DNA Using CRISPR-Cas9 | journal = Frontiers in Cellular and Infection Microbiology | volume = 7 | pages = 91 | date = 2017-03-22 | pmid = 28382278 | pmc = 5360708 | doi = 10.3389/fcimb.2017.00091 | doi-access = free }} Cas9 has been used to repair the mutations causing cataracts in mice.

{{update|how many major types|inaccurate=yes|date=September 2021}}

File:The Stages of CRISPR immunity.svg

CRISPR-Cas systems are divided into three major types (type I, type II, and type III) and twelve subtypes, which are based on their genetic content and structural differences. However, the core defining features of all CRISPR-Cas systems are the cas genes and their proteins: cas1 and cas2 are universal across types and subtypes, while cas3, cas9, and cas10 are signature genes for type I, type II, and type III, respectively.

Adaptation involves recognition and integration of spacers between two adjacent repeats in the CRISPR locus. The "Protospacer" refers to the sequence on the viral genome that corresponds to the spacer. A short stretch of conserved nucleotides exists proximal to the protospacer, which is called the protospacer adjacent motif (PAM). The PAM is a recognition motif that is used to acquire the DNA fragment.{{cite journal | vauthors = Garneau JE, Dupuis MÈ, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadán AH, Moineau S | title = The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA | journal = Nature | volume = 468 | issue = 7320 | pages = 67–71 | date = November 2010 | pmid = 21048762 | doi = 10.1038/nature09523 | s2cid = 205222849 | citeseerx = 10.1.1.451.9645 | bibcode = 2010Natur.468...67G }} In type II, Cas9 recognizes the PAM during adaptation in order to ensure the acquisition of functional spacers.

Loss of spacers and even groups of several have also been observed by Aranaz et al. 2004 and Pourcel et al. 2007. This probably occurs through homologous recombination of the between-repeat material.{{cite journal | vauthors = Deveau H, Garneau JE, Moineau S | title = CRISPR/Cas system and its role in phage-bacteria interactions | journal = Annual Review of Microbiology | volume = 64 | issue = 1 | pages = 475–493 | date = 2010-10-13 | pmid = 20528693 | doi = 10.1146/annurev.micro.112408.134123 | publisher = Annual Reviews }}

CRISPR expression includes the transcription of a primary transcript called a CRISPR RNA (pre-crRNA), which is transcribed from the CRISPR locus by RNA polymerase. Specific endoribonucleases then cleave the pre-crRNAs into small CRISPR RNAs (crRNAs).{{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 }}

Interference involves the crRNAs within a multi-protein complex called CASCADE, which can recognize and specifically base-pair with regions of inserting complementary foreign DNA. The crRNA-foreign nucleic acid complex is then cleaved, however if there are mismatches between the spacer and the target DNA, or if there are mutations in the PAM, then cleavage will not be initiated. In the latter scenario, the foreign DNA is not targeted for attack by the cell, thus the replication of the virus proceeds and the host is not immune to viral infection. The interference stage can be mechanistically and temporally distinct from CRISPR acquisition and expression, yet for complete function as a defense system, all three phases must be functional.{{cite journal | vauthors = Karginov FV, Hannon GJ | title = The CRISPR system: small RNA-guided defense in bacteria and archaea | journal = Molecular Cell | volume = 37 | issue = 1 | pages = 7–19 | date = January 2010 | pmid = 20129051 | pmc = 2819186 | doi = 10.1016/j.molcel.2009.12.033 }}

Stage 1: CRISPR spacer integration. Protospacers and protospacer-associated motifs (shown in red) are acquired at the "leader" end of a CRISPR array in the host DNA. The CRISPR array is composed of spacer sequences (shown in colored boxes) flanked by repeats (black diamonds). This process requires Cas1 and Cas2 (and Cas9 in type II), which are encoded in the cas locus, which are usually located near the CRISPR array.

Stage 2: CRISPR expression. Pre-crRNA is transcribed starting at the leader region by the host RNA polymerase and then cleaved by Cas proteins into smaller crRNAs containing a single spacer and a partial repeat (shown as hairpin structure with colored spacers).

Stage 3: CRISPR interference. crRNA with a spacer that has strong complementarity to the incoming foreign DNA begins a cleavage event (depicted with scissors), which requires Cas proteins. DNA cleavage interferes with viral replication and provides immunity to the host. The interference stage can be functionally and temporarily distinct from CRISPR acquisition and expression (depicted by white line dividing the cell).

dCas9, also referred to as endonuclease deficient Cas9 can be utilized to edit gene expression when applied to the transcription binding site of the desired section of a gene. The optimal function of dCas9 is attributed to its mode of action. Gene expression is inhibited when nucleotides are no longer added to the RNA chain and therefore terminating elongation of that chain, and as a result affects the transcription process. This process occurs when dCas9 is mass-produced so it is able to affect the most genes at any given time via a sequence specific guide RNA molecule. Since dCas9 appears to down regulate gene expression, this action is amplified even more when it is used in conjunction with repressive chromatin modifier domains.{{cite journal | vauthors = Jensen ED, Ferreira R, Jakočiūnas T, Arsovska D, Zhang J, Ding L, Smith JD, David F, Nielsen J, Jensen MK, Keasling JD | title = Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies | journal = Microbial Cell Factories | volume = 16 | issue = 1 | pages = 46 | date = March 2017 | pmid = 28298224 | pmc = 5353793 | doi = 10.1186/s12934-017-0664-2 | doi-access = free }} The dCas9 protein has other functions outside of the regulation of gene expression. A promoter can be added to the dCas9 protein which allows them to work with each other to become efficient at beginning or stopping transcription at different sequences along a strand of DNA. These two proteins are specific in where they act on a gene. This is prevalent in certain types of prokaryotes when a promoter and dCas9 align themselves together to impede the ability of elongation of polymer of nucleotides coming together to form a transcribed piece of DNA. Without the promoter, the dCas9 protein does not have the same effect by itself or with a gene body.{{cite journal | vauthors = Pinto BS, Saxena T, Oliveira R, Méndez-Gómez HR, Cleary JD, Denes LT, McConnell O, Arboleda J, Xia G, Swanson MS, Wang ET | title = Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9 | journal = Molecular Cell | volume = 68 | issue = 3 | pages = 479–490.e5 | date = November 2017 | pmid = 29056323 | pmc = 6013302 | doi = 10.1016/j.molcel.2017.09.033 }}

When examining the effects of repression of transcription further, H3K27, an amino acid component of a histone, becomes methylated through the interaction of dCas9 and a peptide called FOG1. Essentially, this interaction causes gene repression on the C + N terminal section of the amino acid complex at the specific junction of the gene, and as a result, terminates transcription.{{cite journal | vauthors = O'Geen H, Ren C, Nicolet CM, Perez AA, Halmai J, Le VM, Mackay JP, Farnham PJ, Segal DJ | title = dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression | journal = Nucleic Acids Research | volume = 45 | issue = 17 | pages = 9901–9916 | date = September 2017 | pmid = 28973434 | pmc = 5622328 | doi = 10.1093/nar/gkx578 }}

dCas9 also proves to be efficient when it comes to altering certain proteins that can create diseases. When the dCas9 attaches to a form of RNA called guide-RNA, it prevents the proliferation of repeating codons and DNA sequences that might be harmful to an organism's genome. Essentially, when multiple repeat codons are produced, it elicits a response, or recruits an abundance of dCas9 to combat the overproduction of those codons and results in the shut-down of transcription. dCas9 works synergistically with gRNA and directly affects the DNA polymerase II from continuing transcription.

Further explanation of how the dCas9 protein works can be found in their utilization of plant genomes by the regulation of gene production in plants to either increase or decrease certain characteristics. The CRISPR-CAS9 system has the ability to either upregulate or downregulate genes. The dCas9 proteins are a component of the CRISPR-CAS9 system and these proteins can repress certain areas of a plant gene. This happens when dCAS9 binds to repressor domains, and in the case of the plants, deactivation of a regulatory gene such as AtCSTF64, does occur.{{cite book | vauthors = Lowder LG, Paul JW, Qi Y | title = Plant Gene Regulatory Networks | chapter = Multiplexed Transcriptional Activation or Repression in Plants Using CRISPR-dCas9-Based Systems | series = Methods in Molecular Biology | volume = 1629 | pages = 167–184 | date = 2017 | pmid = 28623586 | doi = 10.1007/978-1-4939-7125-1_12 | isbn = 978-1-4939-7124-4 }}

Bacteria are another focus of the usage of dCas9 proteins as well. Since eukaryotes have a larger DNA makeup and genome; the much smaller bacteria are easy to manipulate. As a result, eukaryotes use dCas9 to inhibit RNA polymerase from continuing the process of transcription of genetic material.{{cite journal | vauthors = Barrangou R, Horvath P | title = A decade of discovery: CRISPR functions and applications | journal = Nature Microbiology | volume = 2 | issue = 7 | pages = 17092 | date = June 2017 | pmid = 28581505 | doi = 10.1038/nmicrobiol.2017.92 | s2cid = 19072900 }}

File:Cas9 5AXW.png

Cas9 features a bi-lobed architecture with the guide RNA nestled between the alpha-helical lobe (blue) and the nuclease lobe (cyan, orange, and gray). These two lobes are connected through a single bridge helix. There are two nuclease domains located in the multi-domain nuclease lobe, the RuvC (gray) which cleaves the non-target DNA strand, and the HNH nuclease domain (cyan) that cleaves the target strand of DNA. The RuvC domain is encoded by sequentially disparate sites that interact in the tertiary structure to form the RuvC cleavage domain (See right figure).

File:Cas9 Apo Structure.png

A key feature of the target DNA is that it must contain a protospacer adjacent motif (PAM) consisting of the three-nucleotide sequence- NGG. This PAM is recognized by the PAM-interacting domain (PI domain, orange) located near the C-terminal end of Cas9. Cas9 undergoes distinct conformational changes between the apo, guide RNA bound, and guide RNA:DNA bound states.

Cas9 recognizes the stem-loop architecture inherent in the CRISPR locus, which mediates the maturation of crRNA-tracrRNA ribonucleoprotein complex.{{cite journal | vauthors = Wiedenheft B, Sternberg SH, Doudna JA | title = RNA-guided genetic silencing systems in bacteria and archaea | journal = Nature | volume = 482 | issue = 7385 | pages = 331–338 | date = February 2012 | pmid = 22337052 | doi = 10.1038/nature10886 | s2cid = 205227944 | bibcode = 2012Natur.482..331W }} Cas9 in complex with CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) further recognizes and degrades the target dsDNA.{{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 }} In the co-crystal structure shown here, the crRNA-tracrRNA complex is replaced by a chimeric single-guide RNA (sgRNA, in red) which has been proved to have the same function as the natural RNA complex. The sgRNA base paired with target ssDNA is anchored by Cas9 as a T-shaped architecture. This crystal structure of the DNA-bound Cas9 enzyme reveals distinct conformational changes in the alpha-helical lobe with respect to the nuclease lobe, as well as the location of the HNH domain. The protein consists of a recognition lobe (REC) and a nuclease lobe (NUC). All regions except the HNH form tight interactions with each other and sgRNA-ssDNA complex, while the HNH domain forms few contacts with the rest of the protein. In another conformation of Cas9 complex observed in the crystal, the HNH domain is not visible. These structures suggest the conformational flexibility of HNH domain.

To date, at least three crystal structures have been studied and published. One representing a conformation of Cas9 in the apo state, and two representing Cas9 in the DNA bound state.

File:GRNA-Cas9.svg

In sgRNA-Cas9 complex, based on the crystal structure, REC1, BH and PI domains have important contacts with backbone or bases in both repeat and spacer region.{{cite journal | vauthors = Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O | title = Crystal structure of Cas9 in complex with guide RNA and target DNA | journal = Cell | volume = 156 | issue = 5 | pages = 935–949 | date = February 2014 | pmid = 24529477 | pmc = 4139937 | doi = 10.1016/j.cell.2014.02.001 }}{{cite journal | vauthors = Anders C, Niewoehner O, Duerst A, Jinek M | title = Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease | journal = Nature | volume = 513 | issue = 7519 | pages = 569–573 | date = September 2014 | pmid = 25079318 | pmc = 4176945 | doi = 10.1038/nature13579 | bibcode = 2014Natur.513..569A }} Several Cas9 mutants including REC1 or REC2 domains deletion and residues mutations in BH have been tested. REC1 and BH related mutants show lower or none activity compared with wild type, which indicate these two domains are crucial for the sgRNA recognition at repeat sequence and stabilization of the whole complex. Although the interactions between spacer sequence and Cas9 as well as PI domain and repeat region need further studies, the co-crystal demonstrates clear interface between Cas9 and sgRNA.

File:Cas9 cleavage position.svg

Previous sequence analysis and biochemical studies have posited that Cas9 contains two nuclease domains: an McrA-like HNH nuclease domain and a RuvC-like nuclease domain.{{cite journal | vauthors = Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV | title = A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action | journal = Biology Direct | volume = 1 | issue = 1 | pages = 7 | date = March 2006 | pmid = 16545108 | pmc = 1462988 | doi = 10.1186/1745-6150-1-7 | doi-access = free }} These HNH and RuvC-like nuclease domains are responsible for cleavage of the complementary/target and non-complementary/non-target DNA strands, respectively. Despite low sequence similarity, the sequence similar to RNase H has a RuvC fold (one member of RNase H family) and the HNH region folds as T4 Endo VII (one member of HNH endonuclease family).{{Citation needed|date=December 2019}}

Wild-type S. pyogenes Cas9 requires magnesium (Mg²⁺) cofactors for the RNA-mediated DNA cleavage; however, Cas9 has been shown to exhibit varying levels of activity in the presence of other divalent metal ions. For instance, Cas9 in the presence of manganese (Mn²⁺) has been shown to be capable of RNA-independent DNA cleavage.{{cite journal | vauthors = Sundaresan R, Parameshwaran HP, Yogesha SD, Keilbarth MW, Rajan R | title = RNA-Independent DNA Cleavage Activities of Cas9 and Cas12a | journal = Cell Reports | volume = 21 | issue = 13 | pages = 3728–3739 | date = December 2017 | pmid = 29281823 | pmc = 5760271 | doi = 10.1016/j.celrep.2017.11.100 }} The kinetics of DNA cleavage by Cas9 have been of great interest to the scientific community, as this data provides insight into the intricacies of the reaction. While the cleavage of DNA by RNA-bound Cas9 has been shown to be relatively rapid (k ≥ 700 s⁻¹), the release of the cleavage products is very slow (t_1/2 = ln(2)/k ≈ 43–91 h), essentially rendering Cas9 a single-turnover enzyme.{{cite journal | vauthors = Raper AT, Stephenson AA, Suo Z | title = Functional Insights Revealed by the Kinetic Mechanism of CRISPR/Cas9 | journal = Journal of the American Chemical Society | volume = 140 | issue = 8 | pages = 2971–2984 | date = February 2018 | pmid = 29442507 | doi = 10.1021/jacs.7b13047 | bibcode = 2018JAChS.140.2971R }} Additional studies regarding the kinetics of Cas9 have shown engineered Cas9 to be effective in reducing off-target effects by modifying the rate of the reaction.{{cite journal | vauthors = Lee JK, Jeong E, Lee J, Jung M, Shin E, Kim YH, Lee K, Jung I, Kim D, Kim S, Kim JS | title = Directed evolution of CRISPR-Cas9 to increase its specificity | journal = Nature Communications | volume = 9 | issue = 1 | pages = 3048 | date = August 2018 | pmid = 30082838 | pmc = 6078992 | doi = 10.1038/s41467-018-05477-x | bibcode = 2018NatCo...9.3048L }}{{cite journal | vauthors = Singh D, Wang Y, Mallon J, Yang O, Fei J, Poddar A, Ceylan D, Bailey S, Ha T | title = Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis | journal = Nature Structural & Molecular Biology | volume = 25 | issue = 4 | pages = 347–354 | date = April 2018 | pmid = 29622787 | pmc = 6195204 | doi = 10.1038/s41594-018-0051-7 }}

The cleavage efficiency of Cas9 depends on numerous factors. A key requirement is the presence of a valid PAM at the non-target strand 3 nucleotides downstream from the cleavage site. The canonical PAM sequence for S. Pyogenes Cas9 is NGG, but alternative motifs are tolerated with lower cleavage activity. The most efficient alternative PAM motifs for the wild-type S. Pyogenes Cas9 are NAG and NGA.{{cite journal | vauthors = Kim N, Kim HK, Lee S, Seo JH, Choi JW, Park J, Min S, Yoon S, Cho SR, Kim HH | title = Prediction of the sequence-specific cleavage activity of Cas9 variants | journal = Nature Biotechnology | volume = 38 | issue = 11 | pages = 1328–1336 | date = November 2020 | pmid = 32514125 | doi = 10.1038/s41587-020-0537-9 | s2cid = 219542792 }}{{cite journal | vauthors = Corsi GI, Qu K, Alkan F, Pan X, Luo Y, Gorodkin J | title = CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context | journal = Nature Communications | volume = 13 | issue = 1 | pages = 3006 | date = May 2022 | pmid = 35637227 | pmc = 9151727 | doi = 10.1038/s41467-022-30515-0 | bibcode = 2022NatCo..13.3006C | doi-access = free }} The sequence composition at the target DNA site complementary to the 20 nucleotide spacer region of the gRNA also affects cleavage efficiency. The most relevant nucleotide composition properties that impact efficiency are those in the PAM-proximal region.{{cite journal | vauthors = Xu H, Xiao T, Chen CH, Li W, Meyer CA, Wu Q, Wu D, Cong L, Zhang F, Liu JS, Brown M, Liu XS | title = Sequence determinants of improved CRISPR sgRNA design | journal = Genome Research | volume = 25 | issue = 8 | pages = 1147–1157 | date = August 2015 | pmid = 26063738 | pmc = 4509999 | doi = 10.1101/gr.191452.115 }}{{cite journal | vauthors = Xiang X, Corsi GI, Anthon C, Qu K, Pan X, Liang X, Han P, Dong Z, Liu L, Zhong J, Ma T, Wang J, Zhang X, Jiang H, Xu F, Liu X, Xu X, Wang J, Yang H, Bolund L, Church GM, Lin L, Gorodkin J, Luo Y | title = Enhancing CRISPR-Cas9 gRNA efficiency prediction by data integration and deep learning | journal = Nature Communications | volume = 12 | issue = 1 | pages = 3238 | date = May 2021 | pmid = 34050182 | pmc = 8163799 | doi = 10.1038/s41467-021-23576-0 | bibcode = 2021NatCo..12.3238X | doi-access = free }} Free energy changes of nucleic acids are also highly relevant in defining cleavage activity.{{cite journal | vauthors = Alkan F, Wenzel A, Anthon C, Havgaard JH, Gorodkin J | title = CRISPR-Cas9 off-targeting assessment with nucleic acid duplex energy parameters | journal = Genome Biology | volume = 19 | issue = 1 | pages = 177 | date = October 2018 | pmid = 30367669 | pmc = 6203265 | doi = 10.1186/s13059-018-1534-x | doi-access = free }} In addition to efficiency, the nucleotide composition of the five nucleotides closest to the PAM in the target sequence also affects the scission profile, influencing whether DNA cleavage is blunt or staggered.{{cite journal | vauthors = Longo GM, Sayols S, Kotini AG, Heinen S, Möckel MM, Beli P, Roukos V | title = Linking CRISPR-Cas9 double-strand break profiles to gene editing precision with BreakTag | journal = Nature Biotechnology | date = May 2024 | pmid = 38740992 | doi = 10.1038/s41587-024-02238-8 | doi-access = free | pmc = 11994453 }} Guide RNAs that bind to the DNA forming a duplex that falls into a restricted range of binding free energy changes that excludes extremely weak or stable bindings generally perform efficiently. Stable guide RNA folding conformations can also impair cleavage.{{cite journal | vauthors = Thyme SB, Akhmetova L, Montague TG, Valen E, Schier AF | title = Internal guide RNA interactions interfere with Cas9-mediated cleavage | journal = Nature Communications | volume = 7 | issue = 11750 | pages = 11750 | date = June 2016 | pmid = 27282953 | pmc = 4906408 | doi = 10.1038/ncomms11750 | bibcode = 2016NatCo...711750T }}

The Cas9 nuclease contains two nuclease domains, HNH and RuvC, responsible for cleaving the target strand (TS) and nontarget strand (NTS), respectively. The seminal structural characterization of the nuclease demonstrated that the HNH domain precisely cleaves between the positions 18 and 17 (18|17) of the protospacer, while the RuvC cuts between the same bases and at additional downstream positions.{{cite journal | vauthors = Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E | title = A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity | journal = Science | volume = 337 | issue = 6096 | pages = 816–821 | date = August 2012 | pmid = 22745249 | pmc = 6286148 | doi = 10.1126/science.1225829 }}

Using molecular dynamics simulation, a study reported that cleavage of the NTS between 17|16 of the target sequence was more energetically favored than 18|17, generating 1 nucleotide 5’ ssDNA overhangs.{{cite journal | vauthors = Zuo Z, Liu J | title = Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations | journal = Scientific Reports | volume = 5 | issue = 37584 | pages = 37584 | date = November 2016 | pmid = 27874072 | pmc = 5118739 | doi = 10.1038/srep37584 }} Notably, the authors demonstrated that the 5’ overhangs are filled in, and the product of DNA repair are templated insertions, where the 5’ overhang is used as a template by Pol4 for the repair reaction. The association between staggered cleavage and precise templated insertions was supported by additional studies in human cells.{{cite journal | vauthors = Shi X, Shou J, Mehryar MM, Li J, Wang L, Zhang M, Huang H, Sun X, Wu Q | title = Cas9 has no exonuclease activity resulting in staggered cleavage with overhangs and predictable di- and tri-nucleotide CRISPR insertions without template donor | journal = Cell Discovery | volume = 5 | issue = 53 | pages = 53 | date = October 2019 | pmid = 31636963 | pmc = 6796948 | doi = 10.1038/s41421-019-0120-z }}{{cite journal | vauthors = Shou J, Li J, Liu Y, Wu Q | title = Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-Mediated Nucleotide Insertion | journal = Molecular Cell | volume = 71 | issue = 4 | pages = 498–509.e4 | date = August 2018 | pmid = 30033371 | doi = 10.1016/j.molcel.2018.06.021 | doi-access = free }}

Recently, a high-throughput investigation of Cas9 scission profile revealed that ~85% of on-target cleavage is blunt, whereas ~15% had a 1 nucleotide 5' overhang. Off-targets had a higher staggered cleavage rate compared to on-target sites, with approximately 1/3 of off-targets displaying 5' overhangs from 1 to 3 nucleotides. The scission profile analysis revealed that sequence patterns in the target sequence favor the formation of blunt or staggered DNA cuts, and staggered cleavage favored the formation of predictable indels.

Most archaea and bacteria stubbornly refuse to allow a Cas9 to edit their genome. This is because they can attach foreign DNA, that does not affect them, into their genome. Another way that these cells defy Cas9 is by process of restriction modification (RM) system. When a bacteriophage enters a bacteria or archaea cell it is targeted by the RM system. The RM system then cuts the bacteriophages DNA into separate pieces by restriction enzymes and uses endonucleases to further destroy the strands of DNA. This poses a problem to Cas9 editing because the RM system also targets the foreign genes added by the Cas9 process.{{cite journal | vauthors = Kusano K, Naito T, Handa N, Kobayashi I | title = Restriction-modification systems as genomic parasites in competition for specific sequences | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 92 | issue = 24 | pages = 11095–11099 | date = November 1995 | pmid = 7479944 | pmc = 40578 | doi = 10.1073/pnas.92.24.11095 | doi-access = free | bibcode = 1995PNAS...9211095K }}

Due to the unique ability of Cas9 to bind to essentially any complement sequence in any genome, researchers wanted to use this enzyme to repress transcription of various genomic loci. To accomplish this, the two crucial catalytic residues of the RuvC and HNH domain can be mutated to alanine abolishing all endonuclease activity of Cas9. The resulting protein coined 'dead' Cas9 or 'dCas9' for short, can still tightly bind to dsDNA. This catalytically inactive Cas9 variant has been used for both mechanistic studies into Cas9 DNA interrogative binding and as a general programmable DNA binding RNA-Protein complex.

The interaction of dCas9 with target dsDNA is so tight that high molarity urea protein denaturant can not fully dissociate the dCas9 RNA-protein complex from dsDNA target.{{cite journal | vauthors = Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA | title = DNA interrogation by the CRISPR RNA-guided endonuclease Cas9 | journal = Nature | volume = 507 | issue = 7490 | pages = 62–67 | date = March 2014 | pmid = 24476820 | pmc = 4106473 | doi = 10.1038/nature13011 | bibcode = 2014Natur.507...62S }} dCas9 has been targeted with engineered single guide RNAs to transcription initiation sites of any loci where dCas9 can compete with RNA polymerase at promoters to halt transcription. Also, dCas9 can be targeted to the coding region of loci such that inhibition of RNA Polymerase occurs during the elongation phase of transcription.{{cite journal | vauthors = Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA | title = Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system | journal = Nucleic Acids Research | volume = 41 | issue = 15 | pages = 7429–7437 | date = August 2013 | pmid = 23761437 | pmc = 3753641 | doi = 10.1093/nar/gkt520 }} In Eukaryotes, silencing of gene expression can be extended by targeting dCas9 to enhancer sequences, where dCas9 can block assembly of transcription factors leading to silencing of specific gene expression. Moreover, the guide RNAs provided to dCas9 can be designed to include specific mismatches to its complementary cognate sequence that will quantitatively weaken the interaction of dCas9 for its programmed cognate sequence allowing a researcher to tune the extent of gene silencing applied to a gene of interest.

This technology is similar in principle to RNAi such that gene expression is being modulated at the RNA level. However, the dCas9 approach has gained much traction as there exist less off-target effects and in general larger and more reproducible silencing effects through the use of dCas9 compared to RNAi screens.{{cite journal | vauthors = Heintze J, Luft C, Ketteler R | title = A CRISPR CASe for high-throughput silencing | journal = Frontiers in Genetics | volume = 4 | pages = 193 | year = 2013 | pmid = 24109485 | pmc = 3791873 | doi = 10.3389/fgene.2013.00193 | doi-access = free }} Furthermore, because the dCas9 approach to gene silencing can be quantitatively controlled, a researcher can now precisely control the extent to which a gene of interest is repressed allowing more questions about gene regulation and gene stoichiometry to be answered.

Beyond direct binding of dCas9 to transcriptionally sensitive positions of loci, dCas9 can be fused to a variety of modulatory protein domains to carry out a myriad of functions. Recently, dCas9 has been fused to chromatin remodeling proteins (HDACs/HATs) to reorganize the chromatin structure around various loci. This is important in targeting various eukaryotic genes of interest as heterochromatin structures hinder Cas9 binding. Furthermore, because Cas9 can react to heterochromatin, it is theorized that this enzyme can be further applied to studying the chromatin structure of various loci. Additionally, dCas9 has been employed in genome wide screens of gene repression. By employing large libraries of guide RNAs capable of targeting thousands of genes, genome wide genetic screens using dCas9 have been conducted.{{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 }}

Another method for silencing transcription with Cas9 is to directly cleave mRNA products with the catalytically active Cas9 enzyme.{{cite journal | vauthors = O'Connell MR, Oakes BL, Sternberg SH, East-Seletsky A, Kaplan M, Doudna JA | title = Programmable RNA recognition and cleavage by CRISPR/Cas9 | journal = Nature | volume = 516 | issue = 7530 | pages = 263–266 | date = December 2014 | pmid = 25274302 | pmc = 4268322 | doi = 10.1038/nature13769 | bibcode = 2014Natur.516..263O }} This approach is made possible by hybridizing ssDNA with a PAM complement sequence to ssRNA allowing for a dsDNA-RNA PAM site for Cas9 binding. This technology makes available the ability to isolate endogenous RNA transcripts in cells without the need to induce chemical modifications to RNA or RNA tagging methods.

In contrast to silencing genes, dCas9 can also be used to activate genes when fused to transcription activating factors. These factors include subunits of bacterial RNA Polymerase II and traditional transcription factors in eukaryotes. Recently, genome-wide screens of transcription activation have also been accomplished using dCas9 fusions named 'CRISPRa' for activation.

{{Portal|Biology|Technology}}

• DCas9 activation system
• CRISPR
• CRISPR gene editing
• Genome editing
• Zinc finger nuclease
• Transcription activator-like effector nuclease

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• {{cite journal | vauthors = Kennedy EM, Cullen BR | title = Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment | journal = Virology | volume = 479-480 | pages = 213–220 | date = May 2015 | pmid = 25759096 | pmc = 4424069 | doi = 10.1016/j.virol.2015.02.024 }}
• {{cite journal | vauthors = Abbott TR, Dhamdhere G, Liu Y, Lin X, Goudy L, Zeng L, Chemparathy A, Chmura S, Heaton NS, Debs R, Pande T, Endy D, La Russa MF, Lewis DB, Qi LS | title = Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza | journal = Cell | volume = 181 | issue = 4 | pages = 865–876.e12 | date = May 2020 | pmid = 32353252 | pmc = 7189862 | doi = 10.1016/j.cell.2020.04.020 | doi-access = free }}
• {{cite magazine |author=Steven Levy |date=March 18, 2020 |title=Could Crispr Be Humanity's Next Virus Killer? |magazine=Wired |url=https://www.wired.com/story/could-crispr-be-the-next-virus-killer/}}

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• {{PDBe-KB2|Q99ZW2|Streptococcus pyogenes CRISPR-associated endonuclease Cas9/Csn1}}

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Category:Deoxyribonucleases

Category:Repetitive DNA sequences

Category:Immune system

Category:Bacterial proteins

Category:Genome editing