Editing Functional genomics (section)

===Mutagenesis and phenotyping===
An important functional feature of genes is the phenotype caused by mutations. Mutants can be produced by random mutations or by directed mutagenesis, including site-directed mutagenesis, deleting complete genes, or other techniques.

====Knock-outs (gene deletions)====
Gene function can be investigated by systematically "knocking out" genes one by one. This is done by either [[gene knockout|deletion]] or disruption of function (such as by [[insertional mutagenesis]]) and the resulting organisms are screened for phenotypes that provide clues to the function of the disrupted gene. Knock-outs have been produced for whole genomes, i.e. by deleting all genes in a genome. For [[essential gene]]s, this is not possible, so other techniques are used, e.g. deleting a gene while expressing the gene from a [[plasmid]], using an inducible promoter, so that the level of gene product can be changed at will (and thus a "functional" deletion achieved).

====Site-directed mutagenesis====
[[Site-directed mutagenesis]] is used to mutate specific bases (and thus [[amino acid]]s). This is critical to investigate the function of specific amino acids in a protein, e.g. in the active site of an [[enzyme]].

====RNAi====
{{Main|RNAi}}
[[RNA interference]] (RNAi) methods can be used to transiently silence or knockdown gene expression using ~20 base-pair double-stranded RNA typically delivered by transfection of synthetic ~20-mer short-interfering RNA molecules (siRNAs) or by virally encoded short-hairpin RNAs (shRNAs). RNAi screens, typically performed in cell culture-based assays or experimental organisms (such as ''C. elegans'') can be used to systematically disrupt nearly every gene in a genome or subsets of genes (sub-genomes); possible functions of disrupted genes can be assigned based on observed [[phenotype]]s.

====CRISPR screens====
[[File:Journal.pbio.2006951.g001-B.png|thumb|upright=1.5|An example of a CRISPR loss-of-function screen<ref>{{cite journal |title=Genome-wide CRISPR screens for Shiga toxins and ricin reveal Golgi proteins critical for glycosylation |vauthors=Tian S, Muneeruddin K, Choi MY, Tao L, Bhuiyan RH, Ohmi Y, Furukawa K, Furukawa K, Boland S, Shaffer SA, Adam RM, Dong M |date=27 November 2018 |journal= PLOS Biology |volume=16 |issue=11 |at=e2006951 |doi-access=free |doi=10.1371/journal.pbio.2006951|pmid=30481169 |pmc=6258472 }}</ref>]]
CRISPR-Cas9 has been used to delete genes in a multiplexed manner in cell-lines. Quantifying the amount of guide-RNAs for each gene before and after the experiment can point towards essential genes. If a guide-RNA disrupts an essential gene it will lead to the loss of that cell and hence there will be a depletion of that particular guide-RNA after the screen. In a recent CRISPR-cas9 experiment in mammalian cell-lines, around 2000 genes were found to be essential in multiple cell-lines.<ref>{{cite journal | vauthors = Hart T, Chandrashekhar M, Aregger M, Steinhart Z, Brown KR, MacLeod G, Mis M, Zimmermann M, Fradet-Turcotte A, Sun S, Mero P, Dirks P, Sidhu S, Roth FP, Rissland OS, Durocher D, Angers S, Moffat J | title = High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities | journal = Cell | volume = 163 | issue = 6 | pages = 1515–26 | date = December 2015 | pmid = 26627737 | doi = 10.1016/j.cell.2015.11.015 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F | title = Genome-scale CRISPR-Cas9 knockout screening in human cells | journal = Science | volume = 343 | issue = 6166 | pages = 84–87 | date = January 2014 | pmid = 24336571 | pmc = 4089965 | doi = 10.1126/science.1247005 | bibcode = 2014Sci...343...84S }}</ref> Some of these genes were essential in only one cell-line. Most of genes are part of multi-protein complexes. This approach can be used to identify synthetic lethality by using the appropriate genetic background. CRISPRi and CRISPRa enable loss-of-function and gain-of-function screens in a similar manner. CRISPRi identified ~2100 essential genes in the K562 cell-line.<ref>{{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 = 647–61 | date = October 2014 | pmid = 25307932 | pmc = 4253859 | doi = 10.1016/j.cell.2014.09.029 }}</ref><ref>{{cite journal | vauthors = Horlbeck MA, Gilbert LA, Villalta JE, Adamson B, Pak RA, Chen Y, Fields AP, Park CY, Corn JE, Kampmann M, Weissman JS | title = Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation | journal = eLife | volume = 5 | date = September 2016 | pmid = 27661255 | pmc = 5094855 | doi = 10.7554/eLife.19760 | doi-access = free }}</ref> CRISPR deletion screens have also been used to identify potential regulatory elements of a gene. For example, a technique called ScanDel was published which attempted this approach. The authors deleted regions outside a gene of interest(HPRT1 involved in a Mendelian disorder) in an attempt to identify regulatory elements of this gene.<ref>{{cite journal | vauthors = Gasperini M, Findlay GM, McKenna A, Milbank JH, Lee C, Zhang MD, Cusanovich DA, Shendure J | title = CRISPR/Cas9-Mediated Scanning for Regulatory Elements Required for HPRT1 Expression via Thousands of Large, Programmed Genomic Deletions | journal = American Journal of Human Genetics | volume = 101 | issue = 2 | pages = 192–205 | date = August 2017 | pmid = 28712454 | pmc = 5544381 | doi = 10.1016/j.ajhg.2017.06.010 }}</ref> Gassperini et al. did not identify any distal regulatory elements for HPRT1 using this approach, however such approaches can be extended to other genes of interest.