Essential gene

Two main strategies have been employed to identify essential genes on a genome-wide basis: directed deletion of genes and random mutagenesis using transposons. In the first case, annotated individual genes (or ORFs) are completely deleted from the genome in a systematic way. In transposon-mediated mutagenesis, transposons are randomly inserted in as many positions in a genome as possible, aiming to disrupt the function of the targeted genes (see figure below). Insertion mutants that are still able to survive or grow suggest the transposon inserted in a gene that is not essential for survival. The location of the transposon insertions can be determined through hybridization to microarrays {{cite journal | vauthors = Sassetti CM, Boyd DH, Rubin EJ | title = Genes required for mycobacterial growth defined by high density mutagenesis | journal = Mol Microbiol | volume = 48 | issue = 1 | pages = 77–84| date = 2003 | pmid = 12657046 | doi = 10.1046/j.1365-2958.2003.03425.x | doi-access = free }} or through transposon sequencing . With the development of CRISPR, gene essentiality has also been determined through inhibition of gene expression through CRISPR interference. A summary of such screens is shown in the table.{{cite journal | vauthors = Gerdes S, Edwards R, Kubal M, Fonstein M, Stevens R, Osterman A | title = Essential genes on metabolic maps | journal = Current Opinion in Biotechnology | volume = 17 | issue = 5 | pages = 448–56 | date = October 2006 | pmid = 16978855 | doi = 10.1016/j.copbio.2006.08.006 }}

Table 1. Essential genes in bacteria. Mutagenesis: targeted mutants are gene deletions; random mutants are transposon insertions. Methods: Clones indicate single gene deletions, population indicates whole population mutagenesis, e.g. using transposons. Essential genes from population screens include genes essential for fitness (see text). ORFs: number of all open reading frames in that genome. Notes: (a) mutant collection available; (b) direct essentiality screening method (e.g. via antisense RNA) that does not provide information about nonessential genes. (c) Only partial dataset available. (d) Includes predicted gene essentiality and data compilation from published single-gene essentiality studies. (e) Project in progress. (f) Deduced by comparison of the two gene essentiality datasets obtained independently in the P. aeruginosa strains PA14 and PAO1. (g) The original result of 271 essential genes has been corrected to 261, with 31 genes that were thought to be essential being in fact non-essential whereas 20 novel essential genes have been described since then. (h) Counting genes with essential domains and those that lead to growth-defects when disrupted as essential, and those who lead to growth-advantage when disrupted as non-essential. (i) Involved a fully saturated mutant library of 14 replicates, with 84.3% of possible insertion sites with at least one transposon insertion. (j) Each essential gene has been independently confirmed at least five times.

File:Transposon insertions and next generation sequencing map essential genes in Mycobacterium tuberculosis.png H37Rv as found by using transposons which insert in random positions in the genome. If no transposons are found in a gene, the gene is most likely essential as it cannot tolerate any insertion. In this example, essential heme biosynthetic genes hemA, hemB, hemC, hemD are devoid of insertions. The number of sequence reads (‘‘reads/TA’’) is shown for the indicated region of the H37Rv chromosome. Potential TA dinucleotide insertions sites are indicated. Image from Griffin et al. 2011.]]

On the basis of genome-wide experimental studies and systems biology analysis, an essential gene database has been developed by Kong et al. (2019) for predicting > 4000 bacterial species.{{Cite journal|last1=Kong|first1=Xiangzhen|last2=Zhu|first2=Bin|last3=Stone|first3=Victoria N.|last4=Ge|first4=Xiuchun|last5=El-Rami|first5=Fadi E.|last6=Donghai|first6=Huangfu|last7=Xu|first7=Ping|date=December 2019|title=ePath: an online database towards comprehensive essential gene annotation for prokaryotes|journal=Scientific Reports|language=en|volume=9|issue=1|pages=12949|doi=10.1038/s41598-019-49098-w|issn=2045-2322|pmc=6737131|pmid=31506471|bibcode=2019NatSR...912949K}}

In Saccharomyces cerevisiae (budding yeast) 15-20% of all genes are essential. In Schizosaccharomyces pombe (fission yeast) 4,836 heterozygous deletions covering 98.4% of the 4,914 protein coding open reading frames have been constructed. 1,260 of these deletions turned out to be essential.{{cite journal | vauthors = Kim DU, Hayles J, Kim D, Wood V, Park HO, Won M, Yoo HS, Duhig T, Nam M, Palmer G, Han S, Jeffery L, Baek ST, Lee H, Shim YS, Lee M, Kim L, Heo KS, Noh EJ, Lee AR, Jang YJ, Chung KS, Choi SJ, Park JY, Park Y, Kim HM, Park SK, Park HJ, Kang EJ, Kim HB, Kang HS, Park HM, Kim K, Song K, Song KB, Nurse P, Hoe KL | display-authors = 6 | title = Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe | journal = Nature Biotechnology | volume = 28 | issue = 6 | pages = 617–623 | date = June 2010 | pmid = 20473289 | pmc = 3962850 | doi = 10.1038/nbt.1628 }}

Similar screens are more difficult to carry out in other multicellular organisms, including mammals (as a model for humans), due to technical reasons, and their results are less clear. However, various methods have been developed for the nematode worm C. elegans,{{cite journal | vauthors = Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, Kanapin A, Le Bot N, Moreno S, Sohrmann M, Welchman DP, Zipperlen P, Ahringer J | s2cid = 15745225 | display-authors = 6 | title = Systematic functional analysis of the Caenorhabditis elegans genome using RNAi | journal = Nature | volume = 421 | issue = 6920 | pages = 231–7 | date = January 2003 | pmid = 12529635 | doi = 10.1038/nature01278 | bibcode = 2003Natur.421..231K | hdl = 10261/63159 }} the fruit fly,{{cite journal | vauthors = Spradling AC, Stern D, Beaton A, Rhem EJ, Laverty T, Mozden N, Misra S, Rubin GM | display-authors = 6 | title = The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes | journal = Genetics | volume = 153 | issue = 1 | pages = 135–77 | date = September 1999 | doi = 10.1093/genetics/153.1.135 | pmid = 10471706 | pmc = 1460730 }} and zebrafish{{cite journal | vauthors = Amsterdam A, Nissen RM, Sun Z, Swindell EC, Farrington S, Hopkins N | title = Identification of 315 genes essential for early zebrafish development | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 101 | issue = 35 | pages = 12792–7 | date = August 2004 | pmid = 15256591 | pmc = 516474 | doi = 10.1073/pnas.0403929101 | bibcode = 2004PNAS..10112792A | doi-access = free }} (see table). A recent study of 900 mouse genes concluded that 42% of them were essential although the selected genes were not representative.{{cite journal | vauthors = White JK, Gerdin AK, Karp NA, Ryder E, Buljan M, Bussell JN, Salisbury J, Clare S, Ingham NJ, Podrini C, Houghton R, Estabel J, Bottomley JR, Melvin DG, Sunter D, Adams NC, Tannahill D, Logan DW, Macarthur DG, Flint J, Mahajan VB, Tsang SH, Smyth I, Watt FM, Skarnes WC, Dougan G, Adams DJ, Ramirez-Solis R, Bradley A, Steel KP | display-authors = 6 | title = Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes | journal = Cell | volume = 154 | issue = 2 | pages = 452–64 | date = July 2013 | pmid = 23870131 | pmc = 3717207 | doi = 10.1016/j.cell.2013.06.022 }}

Gene knockout experiments are not possible or at least not ethical in humans. However, natural mutations have led to the identification of mutations that lead to early embryonic or later death. Note that many genes in humans are not absolutely essential for survival but can cause severe disease when mutated. Such mutations are catalogued in the Online Mendelian Inheritance in Man (OMIM) database. In a computational analysis of genetic variation and mutations in 2,472 human orthologs of known essential genes in the mouse, Georgi et al. found strong, purifying selection and comparatively reduced levels of sequence variation, indicating that these human genes are essential too.{{cite journal | vauthors = Georgi B, Voight BF, Bućan M | title = From mouse to human: evolutionary genomics analysis of human orthologs of essential genes | journal = PLOS Genetics | volume = 9 | issue = 5 | pages = e1003484 | date = May 2013 | pmid = 23675308 | pmc = 3649967 | doi = 10.1371/journal.pgen.1003484 | veditors = Flint J | doi-access = free }}

While it may be difficult to prove that a gene is essential in humans, it can be demonstrated that a gene is not essential or not even causing disease. For instance, sequencing the genomes of 2,636 Icelandic citizens and the genotyping of 101,584 additional subjects found 8,041 individuals who had 1 gene completely knocked out (i.e. these people were homozygous for a non-functional gene).{{cite journal | vauthors = Sulem P, Helgason H, Oddson A, Stefansson H, Gudjonsson SA, Zink F, Hjartarson E, Sigurdsson GT, Jonasdottir A, Jonasdottir A, Sigurdsson A, Magnusson OT, Kong A, Helgason A, Holm H, Thorsteinsdottir U, Masson G, Gudbjartsson DF, Stefansson K | s2cid = 205349719 | display-authors = 6 | title = Identification of a large set of rare complete human knockouts | journal = Nature Genetics | volume = 47 | issue = 5 | pages = 448–52 | date = May 2015 | pmid = 25807282 | doi = 10.1038/ng.3243 }} Of the 8,041 individuals with complete knock-outs, 6,885 were estimated to be homozygotes, 1,249 were estimated to be compound heterozygotes (i.e. they had both alleles of a gene knocked out but the two alleles had different mutations). In these individuals, a total of 1,171 of the 19,135 human (RefSeq) genes (6.1%) were completely knocked out. It was concluded that these 1,171 genes are non-essential in humans — at least no associated diseases were reported. Similarly, the exome sequences of 3222 British Pakistani-heritage adults with high parental relatedness revealed 1111 rare-variant homozygous genotypes with predicted loss of gene function (LOF = knockouts) in 781 genes.{{cite journal | vauthors = Narasimhan VM, Hunt KA, Mason D, Baker CL, Karczewski KJ, Barnes MR, Barnett AH, Bates C, Bellary S, Bockett NA, Giorda K, Griffiths CJ, Hemingway H, Jia Z, Kelly MA, Khawaja HA, Lek M, McCarthy S, McEachan R, O'Donnell-Luria A, Paigen K, Parisinos CA, Sheridan E, Southgate L, Tee L, Thomas M, Xue Y, Schnall-Levin M, Petkov PM, Tyler-Smith C, Maher ER, Trembath RC, MacArthur DG, Wright J, Durbin R, van Heel DA | display-authors = 6 | title = Health and population effects of rare gene knockouts in adult humans with related parents | journal = Science | volume = 352 | issue = 6284 | pages = 474–7 | date = April 2016 | pmid = 26940866 | pmc = 4985238 | doi = 10.1126/science.aac8624 | bibcode = 2016Sci...352..474N }} This study found an average of 140 predicted LOF genotypes (per subject), including 16 rare (minor allele frequency <1%) heterozygotes, 0.34 rare homozygotes, 83.2 common heterozygotes and 40.6 common homozygotes. Nearly all rare homozygous LOF genotypes were found within autozygous segments (94.9%). Even though most of these individuals had no obvious health issue arising from their defective genes, it is possible that minor health issues may be found upon more detailed examination.

A summary of essentiality screens is shown in the table below (mostly based on the Database of Essential Genes.{{cite journal | vauthors = Luo H, Lin Y, Gao F, Zhang CT, Zhang R | title = DEG 10, an update of the database of essential genes that includes both protein-coding genes and noncoding genomic elements | journal = Nucleic Acids Research | volume = 42 | issue = Database issue | pages = D574-80 | date = January 2014 | pmid = 24243843 | pmc = 3965060 | doi = 10.1093/nar/gkt1131 }}

Viruses lack many genes necessary for metabolism, forcing them to hijack the host's metabolism. Screens for essential genes have been carried out in a few viruses. For instance, human cytomegalovirus (CMV) was found to have 41 essential, 88 nonessential, and 27 augmenting ORFs (150 total ORFs). Most essential and augmenting genes are located in the central region, and nonessential genes generally cluster near the ends of the viral genome.{{cite journal | vauthors = Yu D, Silva MC, Shenk T | title = Functional map of human cytomegalovirus AD169 defined by global mutational analysis | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 100 | issue = 21 | pages = 12396–401 | date = October 2003 | pmid = 14519856 | pmc = 218769 | doi = 10.1073/pnas.1635160100 | bibcode = 2003PNAS..10012396Y | doi-access = free }}

Tscharke and Dobson (2015) compiled a comprehensive survey of essential genes in Vaccinia Virus and assigned roles to each of the 223 ORFs of the Western Reserve (WR) strain and 207 ORFs of the Copenhagen strain, assessing their role in replication in cell culture. According to their definition, a gene is considered essential (i.e. has a role in cell culture) if its deletion results in a decrease in virus titre of greater than 10-fold in either a single or multiple step growth curve. All genes involved in wrapped virion production, actin tail formation, and extracellular virion release were also considered as essential. Genes that influence plaque size, but not replication were defined as non-essential. By this definition 93 genes are required for Vaccinia Virus replication in cell culture, while 108 and 94 ORFs, from WR and Copenhagen respectively, are non-essential.{{cite journal | vauthors = Dobson BM, Tscharke DC | title = Redundancy complicates the definition of essential genes for vaccinia virus | journal = The Journal of General Virology | volume = 96 | issue = 11 | pages = 3326–37 | date = November 2015 | pmid = 26290187 | pmc = 5972330 | doi = 10.1099/jgv.0.000266 }} Vaccinia viruses with deletions at either end of the genome behaved as expected, exhibiting only mild or host range defects. In contrast, combining deletions at both ends of the genome for VACV strain WR caused a devastating growth defect on all cell lines tested. This demonstrates that single gene deletions are not sufficient to assess the essentiality of genes and that more genes are essential in Vaccinia virus than originally thought.

One of the bacteriophages screened for essential genes includes mycobacteriophage Giles. At least 35 of the 78 predicted Giles genes (45%) are non-essential for lytic growth. 20 genes were found to be essential.{{cite journal | vauthors = Dedrick RM, Marinelli LJ, Newton GL, Pogliano K, Pogliano J, Hatfull GF | title = Functional requirements for bacteriophage growth: gene essentiality and expression in mycobacteriophage Giles | journal = Molecular Microbiology | volume = 88 | issue = 3 | pages = 577–89 | date = May 2013 | pmid = 23560716 | pmc = 3641587 | doi = 10.1111/mmi.12210 }} A major problem with phage genes is that a majority of their genes remain functionally unknown, hence their role is difficult to assess. A screen of Salmonella enterica phage SPN3US revealed 13 essential genes although it remains a bit obscure how many genes were really tested.{{cite journal | vauthors = Thomas JA, Benítez Quintana AD, Bosch MA, Coll De Peña A, Aguilera E, Coulibaly A, Wu W, Osier MV, Hudson AO, Weintraub ST, Black LW | display-authors = 6 | title = Identification of Essential Genes in the Salmonella Phage SPN3US Reveals Novel Insights into Giant Phage Head Structure and Assembly | journal = Journal of Virology | volume = 90 | issue = 22 | pages = 10284–10298 | date = November 2016 | pmid = 27605673 | pmc = 5105663 | doi = 10.1128/JVI.01492-16 }}

In theory, essential genes are qualitative. However, depending on the surrounding environment, certain essential gene mutants may show partial functions, which can be quantitatively determined in some studies. For instance, a particular gene deletion may reduce growth rate (or fertility rate or other characters) to 90% of the wild-type. If there are isozymes or alternative pathways for the essential genes, they can be deleted completely. Using CRISPR interference, the expression of essential genes can be modulated or "tuned", leading to quantitative (or continuous) relationships between the level of gene-expression and the magnitude of fitness cost exhibited by a given mutant.

{{main| Synthetic lethality}}

Two genes are synthetic lethal if neither one is essential but when both are mutated the double-mutant is lethal. Some studies have estimated that the number of synthetic lethal genes may be on the order of 45% of all genes.{{cite journal | vauthors = Pál C, Papp B, Lercher MJ, Csermely P, Oliver SG, Hurst LD | s2cid = 4424895 | title = Chance and necessity in the evolution of minimal metabolic networks | journal = Nature | volume = 440 | issue = 7084 | pages = 667–70 | date = March 2006 | pmid = 16572170 | doi = 10.1038/nature04568 | bibcode = 2006Natur.440..667P }}{{cite book | vauthors = Mori H, Baba T, Yokoyama K, Takeuchi R, Nomura W, Makishi K, Otsuka Y, Dose H, Wanner BL | chapter = Identification of Essential Genes and Synthetic Lethal Gene Combinations in Escherichia coli K-12 | volume = 1279 | pages = 45–65 | year = 2015 | pmid = 25636612 | doi = 10.1007/978-1-4939-2398-4_4 | isbn = 978-1-4939-2397-7 | series = Methods in Molecular Biology | title = Gene Essentiality }}

File:Essential metabolic genes in bacteria.png of different bacteria. The same protein may be essential in one species but not another.]]

Many genes are essential only under certain circumstances. For instance, if the amino acid lysine is supplied to a cell any gene that is required to make lysine is non-essential. However, when there is no lysine supplied, genes encoding enzymes for lysine biosynthesis become essential, as no protein synthesis is possible without lysine.

Streptococcus pneumoniae appears to require 147 genes for growth and survival in saliva,{{cite journal | vauthors = Verhagen LM, de Jonge MI, Burghout P, Schraa K, Spagnuolo L, Mennens S, Eleveld MJ, van der Gaast-de Jongh CE, Zomer A, Hermans PW, Bootsma HJ | title = Genome-wide identification of genes essential for the survival of Streptococcus pneumoniae in human saliva | journal = PLOS ONE | volume = 9 | issue = 2 | pages = e89541 | year = 2014 | pmid = 24586856 | pmc = 3934895 | doi = 10.1371/journal.pone.0089541 | bibcode = 2014PLoSO...989541V | doi-access = free }} more than the 113-133 that have been found in previous studies.

The deletion of a gene may result in death or in a block of cell division. While the latter case may implicate "survival" for some time, without cell division the cell may still die eventually. Similarly, instead of blocked cell division a cell may have reduced growth or metabolism ranging from nearly undetectable to almost normal. Thus, there is gradient from "essential" to completely non-essential, again depending on the condition. Some authors have thus distinguished between genes "essential for survival" and "essential for fitness".

The role of genetic background. Similar to environmental conditions, the genetic background can determine the essentiality of a gene: a gene may be essential in one individual but not another, given his or her genetic background. Gene duplications are one possible explanation (see below).

Metabolic dependency. Genes involved in certain biosynthetic pathways, such as amino acid synthesis, can become non-essential if one or more amino acids are supplied by culture medium or by another organism.{{cite journal | vauthors = D'Souza G, Kost C | title = Experimental Evolution of Metabolic Dependency in Bacteria | journal = PLOS Genetics | volume = 12 | issue = 11 | pages = e1006364 | date = November 2016 | pmid = 27814362 | pmc = 5096674 | doi = 10.1371/journal.pgen.1006364 | doi-access = free }} This is the main reason why many parasites (e.g. Cryptosporidium hominis){{Cite journal|last1=Xu|first1=Ping|last2=Widmer|first2=Giovanni|last3=Wang|first3=Yingping|last4=Ozaki|first4=Luiz S.|last5=Alves|first5=Joao M.|last6=Serrano|first6=Myrna G.|last7=Puiu|first7=Daniela|last8=Manque|first8=Patricio|last9=Akiyoshi|first9=Donna|last10=Mackey|first10=Aaron J.|last11=Pearson|first11=William R.|s2cid=4394344|date=October 2004|title=The genome of Cryptosporidium hominis|journal=Nature|language=en|volume=431|issue=7012|pages=1107–1112|doi=10.1038/nature02977|pmid=15510150|bibcode=2004Natur.431.1107X|issn=0028-0836|doi-access=free}} or endosymbiontic bacteria lost many genes (e.g. Chlamydia). Such genes may be essential but only present in the host organism. For instance, Chlamydia trachomatis cannot synthesize purine and pyrimidine nucleotides de novo, so these bacteria are dependent on the nucleotide biosynthetic genes of the host.{{cite journal | vauthors = Tipples G, McClarty G | title = The obligate intracellular bacterium Chlamydia trachomatis is auxotrophic for three of the four ribonucleoside triphosphates | journal = Molecular Microbiology | volume = 8 | issue = 6 | pages = 1105–14 | date = June 1993 | pmid = 8361355 | doi = 10.1111/j.1365-2958.1993.tb01655.x | s2cid = 46389854 }}

Another kind of metabolic dependency, unrelated to cross-species interactions, can be found when bacteria are grown under specific nutrient conditions. For example, more than 100 genes become essential when Escherichia coli is grown on nutrient-limited media. Specifically, isocitrate dehydrogenase (icd) and citrate synthase (gltA) are two enzymes that are part of the tricarboxylic acid (TCA) cycle. Both genes are essential in M9 minimal media (which provides only the most basic nutrients). However, when the media is supplementing with 2-oxoglutarate or glutamate, these genes are not essential any more.{{Cite journal|last1=Côté|first1=Jean-Philippe|last2=French|first2=Shawn|last3=Gehrke|first3=Sebastian S.|last4=MacNair|first4=Craig R.|last5=Mangat|first5=Chand S.|last6=Bharat|first6=Amrita|last7=Brown|first7=Eric D.|date=2016-11-22|title=The Genome-Wide Interaction Network of Nutrient Stress Genes in Escherichia coli|journal=mBio|volume=7|issue=6|doi=10.1128/mBio.01714-16|issn=2150-7511|pmc=5120140|pmid=27879333}}

Many genes are duplicated within a genome and many organisms have different metabolic pathways (alternative metabolic pathway) to synthesis same products. Such duplications (paralogs) and alternative metabolic pathways often render essential genes non-essential because the duplicate can replace the original copy. For instance, the gene encoding the enzyme aspartokinase is essential in E. coli. By contrast, the Bacillus subtilis genome contains three copies of this gene, none of which is essential on its own. However, a triple-deletion of all three genes is lethal. In such cases, the essentiality of a gene or a group of paralogs can often be predicted based on the essentiality of an essential single gene in a different species. In yeast, few of the essential genes are duplicated within the genome: 8.5% of the non-essential genes, but only 1% of the essential genes have a homologue in the yeast genome.

In the worm C. elegans, non-essential genes are highly over-represented among duplicates, possibly because duplication of essential genes causes overexpression of these genes. Woods et al. found that non-essential genes are more often successfully duplicated (fixed) and lost compared to essential genes. By contrast, essential genes are less often duplicated but upon successful duplication are maintained over longer periods.{{cite journal | vauthors = Woods S, Coghlan A, Rivers D, Warnecke T, Jeffries SJ, Kwon T, Rogers A, Hurst LD, Ahringer J | display-authors = 6 | title = Duplication and retention biases of essential and non-essential genes revealed by systematic knockdown analyses | journal = PLOS Genetics | volume = 9 | issue = 5 | pages = e1003330 | date = May 2013 | pmid = 23675306 | pmc = 3649981 | doi = 10.1371/journal.pgen.1003330 | veditors = Sternberg PW | doi-access = free }}

File:Conservation of essential genes.png, adapted from ]]

In bacteria, essential genes appear to be more conserved than nonessential genes {{cite journal | vauthors = Jordan IK, Rogozin IB, Wolf YI, Koonin EV | title = Essential genes are more evolutionarily conserved than are nonessential genes in bacteria | journal = Genome Research | volume = 12 | issue = 6 | pages = 962–8 | date = June 2002 | pmid = 12045149 | pmc = 1383730 | doi = 10.1101/gr.87702 }} but the correlation is not very strong. For instance, only 34% of the B. subtilis essential genes have reliable orthologs in all Bacillota and 61% of the E. coli essential genes have reliable orthologs in all Gamma-proteobacteria. Fang et al. (2005) defined persistent genes as the genes present in more than 85% of the genomes of the clade.{{cite journal | vauthors = Fang G, Rocha E, Danchin A | title = How essential are nonessential genes? | journal = Molecular Biology and Evolution | volume = 22 | issue = 11 | pages = 2147–56 | date = November 2005 | pmid = 16014871 | doi = 10.1093/molbev/msi211 | doi-access = }} They found 475 and 611 of such genes for B. subtilis and E. coli, respectively. Furthermore, they classified genes into five classes according to persistence and essentiality: persistent genes, essential genes, persistent nonessential (PNE) genes (276 in B. subtilis, 409 in E. coli), essential nonpersistent (ENP) genes (73 in B. subtilis, 33 in E. coli), and nonpersistent nonessential (NPNE) genes (3,558 in B. subtilis, 3,525 in E. coli). Fang et al. found 257 persistent genes, which exist both in B. subtilis (for the Bacillota) and E. coli (for the Gamma-proteobacteria). Among these, 144 (respectively 139) were previously identified as essential in B. subtilis (respectively E. coli) and 25 (respectively 18) of the 257 genes are not present in the 475 B. subtilis (respectively 611 E. coli) persistent genes. All the other members of the pool are PNE genes.

In eukaryotes, 83% of the one-to-one orthologs between Schizosaccharomyces pombe and Saccharomyces cerevisiae have conserved essentiality, that is, they are nonessential in both species or essential in both species. The remaining 17% of genes are nonessential in one species and essential in the other.{{cite journal | vauthors = Ryan CJ, Krogan NJ, Cunningham P, Cagney G | title = All or nothing: protein complexes flip essentiality between distantly related eukaryotes | journal = Genome Biology and Evolution | volume = 5 | issue = 6 | pages = 1049–59 | year = 2013 | pmid = 23661563 | pmc = 3698920 | doi = 10.1093/gbe/evt074 }} This is quite remarkable, given that S. pombe is separated from S. cerevisiae by approximately 400 million years of evolution.{{cite journal | vauthors = Sipiczki M | title = Where does fission yeast sit on the tree of life? | journal = Genome Biology | volume = 1 | issue = 2 | pages = REVIEWS1011 | year = 2000 | pmid = 11178233 | pmc = 138848 | doi = 10.1186/gb-2000-1-2-reviews1011 | doi-access = free }}

In general, highly conserved and thus older genes (i.e. genes with earlier phylogenetic origin) are more likely to be essential than younger genes - even if they have been duplicated.{{cite journal | vauthors = Chen WH, Trachana K, Lercher MJ, Bork P | title = Younger genes are less likely to be essential than older genes, and duplicates are less likely to be essential than singletons of the same age | journal = Molecular Biology and Evolution | volume = 29 | issue = 7 | pages = 1703–6 | date = July 2012 | pmid = 22319151 | pmc = 3375470 | doi = 10.1093/molbev/mss014 }}

The experimental study of essential genes is limited by the fact that, by definition, inactivation of an essential gene is lethal to the organism. Therefore, they cannot be simply deleted or mutated to analyze the resulting phenotypes (a common technique in genetics).

There are, however, some circumstances in which essential genes can be manipulated. In diploid organisms, only a single functional copy of some essential genes may be needed (haplosufficiency), with the heterozygote displaying an instructive phenotype. Some essential genes can tolerate mutations that are deleterious, but not wholly lethal, since they do not completely abolish the gene's function.

Computational analysis can reveal many properties of proteins without analyzing them experimentally, e.g. by looking at homologous proteins, function, structure etc. (see also below, Predicting essential genes). The products of essential genes can also be studied when expressed in other organisms, or when purified and studied in vitro.

Conditionally essential genes are easier to study. Temperature-sensitive variants of essential genes have been identified which encode products that lose function at high temperatures, and so only show a phenotype at increased temperature.{{cite journal | vauthors = Kofoed M, Milbury KL, Chiang JH, Sinha S, Ben-Aroya S, Giaever G, Nislow C, Hieter P, Stirling PC | display-authors = 6 | title = An Updated Collection of Sequence Barcoded Temperature-Sensitive Alleles of Yeast Essential Genes | journal = G3 | volume = 5 | issue = 9 | pages = 1879–87 | date = July 2015 | pmid = 26175450 | pmc = 4555224 | doi = 10.1534/g3.115.019174 }}

If screens for essential genes are repeated in independent laboratories, they often result in different gene lists. For instance, screens in E. coli have yielded from ~300 to ~600 essential genes (see Table 1). Such differences are even more pronounced when different bacterial strains are used (see Figure 2). A common explanation is that the experimental conditions are different or that the nature of the mutation may be different (e.g. a complete gene deletion vs. a transposon mutant). Transposon screens in particular are hard to reproduce, given that a transposon can insert at many positions within a gene. Insertions towards the 3' end of an essential gene may not have a lethal phenotype (or no phenotype at all) and thus may not be recognized as such. This can lead to erroneous annotations (here: false negatives).{{cite journal | vauthors = Deng J, Su S, Lin X, Hassett DJ, Lu LJ | title = A statistical framework for improving genomic annotations of prokaryotic essential genes | journal = PLOS ONE | volume = 8 | issue = 3 | pages = e58178 | year = 2013 | pmid = 23520492 | pmc = 3592911 | doi = 10.1371/journal.pone.0058178 | bibcode = 2013PLoSO...858178D | veditors = Kim PM | doi-access = free }}

Comparison of CRISPR/cas9 and RNAi screens. Screens to identify essential genes in the human chronic myelogenous leukemia cell line K562 with these two methods showed only limited overlap. At a 10% false positive rate there were ~4,500 genes identified in the Cas9 screen versus ~3,100 in the shRNA screen, with only ~1,200 genes identified in both.{{cite journal | vauthors = Morgens DW, Deans RM, Li A, Bassik MC | title = Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes | journal = Nature Biotechnology | volume = 34 | issue = 6 | pages = 634–6 | date = June 2016 | pmid = 27159373 | pmc = 4900911 | doi = 10.1038/nbt.3567 }}

Different organisms may have different essential genes. For instance, Bacillus subtilis has 271 essential genes. About one-half (150) of the orthologous genes in E. coli are also essential. Another 67 genes that are essential in E. coli are not essential in B. subtilis, while 86 E. coli essential genes have no B. subtilis ortholog. In Mycoplasma genitalium at least 18 genes are essential that are not essential in M. bovis.{{cite journal | vauthors = Sharma S, Markham PF, Browning GF | title = Genes found essential in other mycoplasmas are dispensable in Mycoplasma bovis | journal = PLOS ONE | volume = 9 | issue = 6 | pages = e97100 | year = 2014 | pmid = 24897538 | pmc = 4045577 | doi = 10.1371/journal.pone.0097100 | bibcode = 2014PLoSO...997100S | doi-access = free }} Many of these different essential genes are caused by paralogs or alternative metabolic pathways.

Such different essential genes in bacteria can be used to develop targeted antibacterial therapies against certain specific pathogens to reduce antibiotic resistance in the microbiome era.{{cite journal | vauthors = Stone VN, Xu P | title = Targeted antimicrobial therapy in the microbiome era | journal = Molecular Oral Microbiology | volume = 32 | issue = 6 | pages = 446–454 | date = December 2017 | pmid = 28609586 | pmc = 5697594 | doi = 10.1111/omi.12190 }} Stone et al (2015) have used the difference in essential genes in bacteria to develop selective drugs against the oral pathogen Porphyromonas gingivalis, rather than the beneficial bacteria Streptococcus sanguis.{{cite journal | vauthors = Stone VN, Parikh HI, El-rami F, Ge X, Chen W, Zhang Y, Kellogg GE, Xu P | display-authors = 6 | title = Identification of Small-Molecule Inhibitors against Meso-2, 6-Diaminopimelate Dehydrogenase from Porphyromonas gingivalis | journal = PLOS ONE | volume = 10 | issue = 11 | pages = e0141126 | date = 2015-11-06 | pmid = 26544875 | pmc = 4636305 | doi = 10.1371/journal.pone.0141126 | bibcode = 2015PLoSO..1041126S | veditors = Merritt J | doi-access = free }}

Essential genes can be predicted computationally. However, most methods use experimental data ("training sets") to some extent. Chen et al.{{cite journal | vauthors = Cheng J, Xu Z, Wu W, Zhao L, Li X, Liu Y, Tao S | title = Training set selection for the prediction of essential genes | journal = PLOS ONE | volume = 9 | issue = 1 | pages = e86805 | year = 2014 | pmid = 24466248 | pmc = 3899339 | doi = 10.1371/journal.pone.0086805 | bibcode = 2014PLoSO...986805C | doi-access = free }} determined four criteria to select training sets for such predictions: (1) essential genes in the selected training set should be reliable; (2) the growth conditions in which essential genes are defined should be consistent in training and prediction sets; (3) species used as training set should be closely related to the target organism; and (4) organisms used as training and prediction sets should exhibit similar phenotypes or lifestyles. They also found that the size of the training set should be at least 10% of the total genes to yield accurate predictions. Some approaches for predicting essential genes are:

Comparative genomics. Shortly after the first genomes (of Haemophilus influenzae and Mycoplasma genitalium) became available, Mushegian et al.{{cite journal | vauthors = Mushegian AR, Koonin EV | title = A minimal gene set for cellular life derived by comparison of complete bacterial genomes | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 93 | issue = 19 | pages = 10268–73 | date = September 1996 | pmid = 8816789 | pmc = 38373 | doi = 10.1073/pnas.93.19.10268 | bibcode = 1996PNAS...9310268M | doi-access = free }} tried to predict the number of essential genes based on common genes in these two species. It was surmised that only essential genes should be conserved over the long evolutionary distance that separated the two bacteria. This study identified approximately 250 candidate essential genes. As more genomes became available the number of predicted essential genes kept shrinking because more genomes shared fewer and fewer genes. As a consequence, it was concluded that the universal conserved core consists of less than 40 genes.{{cite journal | vauthors = Charlebois RL, Doolittle WF | title = Computing prokaryotic gene ubiquity: rescuing the core from extinction | journal = Genome Research | volume = 14 | issue = 12 | pages = 2469–77 | date = December 2004 | pmid = 15574825 | pmc = 534671 | doi = 10.1101/gr.3024704 }}{{cite journal | vauthors = Juhas M, Eberl L, Glass JI | title = Essence of life: essential genes of minimal genomes | journal = Trends in Cell Biology | volume = 21 | issue = 10 | pages = 562–8 | date = October 2011 | pmid = 21889892 | doi = 10.1016/j.tcb.2011.07.005 }} However, this set of conserved genes is not identical to the set of essential genes as different species rely on different essential genes.

A similar approach has been used to infer essential genes from the pan-genome of Brucella species. 42 complete Brucella genomes and a total of 132,143 protein-coding genes were used to predict 1252 potential essential genes, derived from the core genome by comparison with a prokaryote database of essential genes.{{cite journal | vauthors = Yang X, Li Y, Zang J, Li Y, Bie P, Lu Y, Wu Q | s2cid = 14565579 | title = Analysis of pan-genome to identify the core genes and essential genes of Brucella spp | journal = Molecular Genetics and Genomics | volume = 291 | issue = 2 | pages = 905–12 | date = April 2016 | pmid = 26724943 | doi = 10.1007/s00438-015-1154-z }}

Network analysis. After the first protein interaction networks of yeast had been published,{{cite journal | vauthors = Schwikowski B, Uetz P, Fields S | s2cid = 3009359 | title = A network of protein-protein interactions in yeast | journal = Nature Biotechnology | volume = 18 | issue = 12 | pages = 1257–61 | date = December 2000 | pmid = 11101803 | doi = 10.1038/82360 }} it was found that highly connected proteins (e.g. by protein-protein interactions) are more likely to be essential.{{cite journal | vauthors = Jeong H, Mason SP, Barabási AL, Oltvai ZN | s2cid = 258942 | title = Lethality and centrality in protein networks | journal = Nature | volume = 411 | issue = 6833 | pages = 41–2 | date = May 2001 | pmid = 11333967 | doi = 10.1038/35075138 | arxiv = cond-mat/0105306 | bibcode = 2001Natur.411...41J }} However, highly connected proteins may be experimental artifacts and high connectivity may rather represent pleiotropy instead of essentiality.{{cite journal | vauthors = Yu H, Braun P, Yildirim MA, Lemmens I, Venkatesan K, Sahalie J, Hirozane-Kishikawa T, Gebreab F, Li N, Simonis N, Hao T, Rual JF, Dricot A, Vazquez A, Murray RR, Simon C, Tardivo L, Tam S, Svrzikapa N, Fan C, de Smet AS, Motyl A, Hudson ME, Park J, Xin X, Cusick ME, Moore T, Boone C, Snyder M, Roth FP, Barabási AL, Tavernier J, Hill DE, Vidal M | display-authors = 6 | title = High-quality binary protein interaction map of the yeast interactome network | journal = Science | volume = 322 | issue = 5898 | pages = 104–10 | date = October 2008 | pmid = 18719252 | pmc = 2746753 | doi = 10.1126/science.1158684 | bibcode = 2008Sci...322..104Y }} Nevertheless, network methods have been improved by adding other criteria and therefore do have some value in predicting essential genes.{{cite journal | vauthors = Li X, Li W, Zeng M, Zheng R, Li M | title = Network-based methods for predicting essential genes or proteins: a survey | journal = Briefings in Bioinformatics | date = February 2019 | volume = 21 | issue = 2 | pages = 566–583 | pmid = 30776072 | doi = 10.1093/bib/bbz017 }}

Machine Learning. Hua et al. used Machine Learning to predict essential genes in 25 bacterial species.{{cite journal | vauthors = Hua HL, Zhang FZ, Labena AA, Dong C, Jin YT, Guo FB | title = An Approach for Predicting Essential Genes Using Multiple Homology Mapping and Machine Learning Algorithms | journal = BioMed Research International | volume = 2016 | pages = 7639397 | date = 2016-01-01 | pmid = 27660763 | pmc = 5021884 | doi = 10.1155/2016/7639397 | doi-access = free }}

Hurst index. Liu et al. (2015){{cite journal | vauthors = Liu X, Wang B, Xu L | title = Statistical Analysis of Hurst Exponents of Essential/Nonessential Genes in 33 Bacterial Genomes | journal = PLOS ONE | volume = 10 | issue = 6 | pages = e0129716 | year = 2015 | pmid = 26067107 | pmc = 4466317 | doi = 10.1371/journal.pone.0129716 | bibcode = 2015PLoSO..1029716L | doi-access = free }} used the Hurst exponent, a characteristic parameter to describe long-range correlation in DNA to predict essential genes. In 31 out of 33 bacterial genomes the significance levels of the Hurst exponents of the essential genes were significantly higher than for the corresponding full-gene-set, whereas the significance levels of the Hurst exponents of the nonessential genes remained unchanged or increased only slightly.

Minimal genomes. It was also thought that essential genes could be inferred from minimal genomes which supposedly contain only essential genes. The problem here is that the smallest genomes belong to parasitic (or symbiontic) species which can survive with a reduced gene set as they obtain many nutrients from their hosts. For instance, one of the smallest genomes is that of Hodgkinia cicadicola, a symbiont of cicadas, containing only 144 Kb of DNA encoding only 188 genes.{{cite journal | vauthors = McCutcheon JP, McDonald BR, Moran NA | title = Origin of an alternative genetic code in the extremely small and GC-rich genome of a bacterial symbiont | journal = PLOS Genetics | volume = 5 | issue = 7 | pages = e1000565 | date = July 2009 | pmid = 19609354 | pmc = 2704378 | doi = 10.1371/journal.pgen.1000565 | veditors = Matic I | doi-access = free }} Like other symbionts, Hodgkinia receives many of its nutrients from its host, so its genes do not need to be essential.

Metabolic modelling. Essential genes may be also predicted in completely sequenced genomes by metabolic reconstruction, that is, by reconstructing the complete metabolism from the gene content and then identifying those genes and pathways that have been found to be essential in other species. However, this method can be compromised by proteins of unknown function. In addition, many organisms have backup or alternative pathways which have to be taken into account (see figure 1). Metabolic modeling was also used by Basler (2015) to develop a method to predict essential metabolic genes.{{cite book | vauthors = Basler G | chapter = Computational Prediction of Essential Metabolic Genes Using Constraint-Based Approaches | volume = 1279 | pages = 183–204 | year = 2015 | pmid = 25636620 | doi = 10.1007/978-1-4939-2398-4_12 | isbn = 978-1-4939-2397-7 | series = Methods in Molecular Biology | title = Gene Essentiality }} Flux balance analysis, a method of metabolic modeling, has recently been used to predict essential genes in clear cell renal cell carcinoma metabolism.{{cite journal | vauthors = Gatto F, Miess H, Schulze A, Nielsen J | title = Flux balance analysis predicts essential genes in clear cell renal cell carcinoma metabolism | journal = Scientific Reports | volume = 5 | pages = 10738 | date = June 2015 | pmid = 26040780 | pmc = 4603759 | doi = 10.1038/srep10738 | bibcode = 2015NatSR...510738G }}

Genes of unknown function. Surprisingly, a significant number of essential genes has no known function. For instance, among the 385 essential candidates in M. genitalium, no function could be ascribed to 95 genes even though this number had been reduced to 75 by 2011. Most of unknown functionally essential genes have potential biological functions related to one of the three fundamental functions.

ZUPLS. Song et al. presented a novel method to predict essential genes that only uses the Z-curve and other sequence-based features.{{cite journal | vauthors = Song K, Tong T, Wu F | title = Predicting essential genes in prokaryotic genomes using a linear method: ZUPLS | journal = Integrative Biology | volume = 6 | issue = 4 | pages = 460–9 | date = April 2014 | pmid = 24603751 | doi = 10.1039/c3ib40241j | doi-access = free }} Such features can be calculated readily from the DNA/amino acid sequences. However, the reliability of this method remains a bit obscure.

Essential gene prediction servers. Guo et al. (2015) have developed three online services to predict essential genes in bacterial genomes. These freely available tools are applicable for single gene sequences without annotated functions, single genes with definite names, and complete genomes of bacterial strains.{{cite book | vauthors = Guo FB, Ye YN, Ning LW, Wei W | chapter = Three Computational Tools for Predicting Bacterial Essential Genes | volume = 1279 | pages = 205–17 | year = 2015 | pmid = 25636621 | doi = 10.1007/978-1-4939-2398-4_13 | isbn = 978-1-4939-2397-7 | series = Methods in Molecular Biology | title = Gene Essentiality }} Kong et al. (2019) have developed the [https://www.pubapps.vcu.edu/epath/ ePath] database, which can be used to search > 4000 bacterial species for predicting essential genes.

Although most essential genes encode proteins, many essential proteins consist of a single domain. This fact has been used to identify essential protein domains. Goodacre et al. have identified hundreds of essential domains of unknown function (eDUFs).{{cite journal | vauthors = Goodacre NF, Gerloff DL, Uetz P | title = Protein domains of unknown function are essential in bacteria | journal = mBio | volume = 5 | issue = 1 | pages = e00744-13 | date = December 2013 | pmid = 24381303 | pmc = 3884060 | doi = 10.1128/mBio.00744-13 }} Lu et al.{{cite book | vauthors = Lu Y, Lu Y, Deng J, Lu H, Lu LJ | chapter = Discovering Essential Domains in Essential Genes | volume = 1279 | pages = 235–45 | year = 2015 | pmid = 25636623 | doi = 10.1007/978-1-4939-2398-4_15 | isbn = 978-1-4939-2397-7 | series = Methods in Molecular Biology | title = Gene Essentiality }} presented a similar approach and identified 3,450 domains that are essential in at least one microbial species.

• Essential amino acid
• Essential proteins in protein complexes
• Gene
• Genome
• Minimal genome
• Mutation

{{Reflist|30em}}

{{refbegin}}

• {{cite book | vauthors = Gao F, Luo H, Zhang CT, Zhang R | chapter = Gene Essentiality Analysis Based on DEG 10, an Updated Database of Essential Genes | volume = 1279 | pages = 219–33 | year = 2015 | pmid = 25636622 | doi = 10.1007/978-1-4939-2398-4_14 | isbn = 978-1-4939-2397-7 | series = Methods in Molecular Biology | title = Gene Essentiality }}
• {{cite book | veditors = Long JL | date = 2015 | title = Gene Essentiality - Springer Methods and Protocols | series = Methods in Molecular Biology | volume = 1279 | publisher = Humana Press | pages = 248 | isbn = 978-1-4939-2397-7| doi = 10.1007/978-1-4939-2398-4 | s2cid = 27547825 }}
• {{cite book | veditors = Zhang R| date = 2022 | title = Essential Genes and Genomes | series = Methods in Molecular Biology | volume = 2377 | publisher = Humana Press | pages = 434 | isbn = 978-1-0716-1719-9| doi = 10.1007/978-1-0716-1720-5| s2cid = 240006552 }}

{{refend}}

• [http://tubic.tju.edu.cn/deg Database of Essential Genes]
• [http://ogeedb.embl.de/#summary OGEE: Online Essentiality Database]
• [https://web.archive.org/web/20151014215511/http://theseed.uchicago.edu/FIG/eggs.cgi EGGS (Essential Genes on Genome Scale) database]
• [https://www.pubapps.vcu.edu/epath/ ePath (Essential genes in pathway) database]
• [http://ecoliwiki.net/colipedia/index.php/Essential_genes Essential genes in E. coli (EcoliWiki])
• [https://web.archive.org/web/20140219171548/http://www.ecogene.org/old/topic.php?topic_id=5 Essential genes in E. coli (Ecogene)]
• Benjamin Lewin's [https://www.pearson.ch/HigherEducation/Pearson/EAN/9780132016360/Essential-Genes-International-Edition Essential Genes] (textbook), Pearson/Prentice-Hall.

Category:Genomics