Editing Functional genomics (section)

==Techniques and applications==
Functional genomics includes function-related aspects of the genome itself such as [[mutation]] and [[polymorphism (biology)|polymorphism]] (such as [[single nucleotide polymorphism]] (SNP) analysis), as well as the measurement of molecular activities. The latter comprise a number of "-[[omics]]" such as [[transcriptomics]] ([[gene expression]]), [[proteomics]] ([[protein production]]), and [[metabolomics]]. Functional genomics uses mostly [[multiplex (assay)|multiplex]] techniques to measure the abundance of many or all gene products such as [[Messenger RNA|mRNA]]s or [[protein]]s within a [[Biological specimen|biological sample]]. A more focused functional genomics approach might test the function of all variants of one gene and quantify the effects of mutants by using sequencing as a readout of activity. Together these measurement modalities endeavor to quantitate the various biological processes and improve our understanding of gene and protein functions and interactions.

===At the DNA level===

====Genetic interaction mapping====
{{Main|Epistasis}}
Systematic pairwise deletion of genes or inhibition of gene expression can be used to identify genes with related function, even if they do not interact physically. [[Epistasis]] refers to the fact that effects for two different gene knockouts may not be additive; that is, the phenotype that results when two genes are inhibited may be different from the sum of the effects of single knockouts.

====DNA/Protein interactions====
{{Main|ChIP sequencing}}
Proteins formed by the translation of the mRNA (messenger RNA, a coded information from DNA for protein synthesis) play a major role in regulating gene expression. To understand how they regulate gene expression it is necessary to identify DNA sequences that they interact with. Techniques have been developed to identify sites of DNA-protein interactions. These include [[ChIP-sequencing]], [[CUT&RUN sequencing]] and Calling Cards.<ref>{{cite journal | vauthors = Wang H, Mayhew D, Chen X, Johnston M, Mitra RD | title = Calling Cards enable multiplexed identification of the genomic targets of DNA-binding proteins | journal = Genome Research | volume = 21 | issue = 5 | pages = 748–55 | date = May 2011 | pmid = 21471402 | doi = 10.1101/gr.114850.110 | pmc = 3083092 }}</ref>

====DNA accessibility assays====
Assays have been developed to identify regions of the genome that are accessible. These regions of accessible chromatin are candidate regulatory regions. These assays include [[ATAC-seq]], [[DNase-Seq]] and [[FAIRE-Seq]].

===At the RNA level===

====Microarrays====
{{Main|DNA microarray}}
[[File:DNA microarray.svg|thumb|A [[DNA microarray]]]]
Microarrays measure the amount of mRNA in a sample that corresponds to a given gene or probe DNA sequence. Probe sequences are immobilized on a solid surface and allowed to [[Nucleic acid hybridization|hybridize]] with fluorescently labeled "target" mRNA. The intensity of fluorescence of a spot is proportional to the amount of target sequence that has hybridized to that spot and therefore to the abundance of that mRNA sequence in the sample. Microarrays allow for the identification of candidate genes involved in a given process based on variation between transcript levels for different conditions and shared expression patterns with genes of known function.

====SAGE====
{{Main|Serial analysis of gene expression}}
[[Serial analysis of gene expression]] (SAGE) is an alternate method of analysis based on RNA sequencing rather than hybridization. SAGE relies on the sequencing of 10–17 base pair tags which are unique to each gene. These tags are produced from [[Polyadenylation|poly-A mRNA]] and ligated end-to-end before sequencing. SAGE gives an unbiased measurement of the number of transcripts per cell, since it does not depend on prior knowledge of what transcripts to study (as microarrays do).

====RNA sequencing====
{{Main|RNA-Seq|MicroRNA sequencing}}
RNA sequencing has taken over microarray and SAGE technology in recent years, as noted in 2016, and has become the most efficient way to study  transcription and gene expression. This is typically done by [[DNA sequencing|next-generation sequencing]].<ref>{{cite journal | vauthors = Hrdlickova R, Toloue M, Tian B | title = RNA-Seq methods for transcriptome analysis | journal = Wiley Interdisciplinary Reviews: RNA | volume = 8 | issue = 1 | pages = e1364 | date = January 2017 | pmid = 27198714 | pmc = 5717752 | doi = 10.1002/wrna.1364 }}</ref>

A subset of sequenced RNAs are small RNAs, a class of non-coding RNA molecules that are key regulators of transcriptional and post-transcriptional gene silencing, or [[RNA silencing]]. Next-generation sequencing is the gold standard tool for [[non-coding RNA]] discovery, profiling and expression analysis.

====Massively Parallel Reporter Assays (MPRAs)====
Massively parallel reporter assays is a technology to test the cis-regulatory activity of DNA sequences.<ref>{{cite journal | vauthors = Kwasnieski JC, Fiore C, Chaudhari HG, Cohen BA | title = High-throughput functional testing of ENCODE segmentation predictions | journal = Genome Research | volume = 24 | issue = 10 | pages = 1595–602 | date = October 2014 | pmid = 25035418 | pmc = 4199366 | doi = 10.1101/gr.173518.114 }}</ref><ref>{{cite journal | vauthors = Patwardhan RP, Hiatt JB, Witten DM, Kim MJ, Smith RP, May D, Lee C, Andrie JM, Lee SI, Cooper GM, Ahituv N, Pennacchio LA, Shendure J | title = Massively parallel functional dissection of mammalian enhancers in vivo | journal = Nature Biotechnology | volume = 30 | issue = 3 | pages = 265–70 | date = February 2012 | pmid = 22371081 | pmc = 3402344 | doi = 10.1038/nbt.2136 }}</ref> MPRAs use a [[plasmid]] with a synthetic cis-regulatory element upstream of a promoter driving a synthetic gene such as Green Fluorescent Protein. A library of cis-regulatory elements is usually tested using MPRAs, a library can contain from hundreds to thousands of cis-regulatory elements. The cis-regulatory activity of the elements is assayed by using the downstream reporter activity. The activity of all the library members is assayed in parallel using barcodes for each cis-regulatory element. One limitation of MPRAs is that the activity is assayed on a plasmid and may not capture all aspects of gene regulation observed in the genome.

====STARR-seq====
{{Main|STARR-seq}}
STARR-seq is a technique similar to MPRAs to assay enhancer activity of randomly sheared genomic fragments. In the original publication,<ref>{{cite journal | vauthors = Arnold CD, Gerlach D, Stelzer C, Boryń ŁM, Rath M, Stark A | title = Genome-wide quantitative enhancer activity maps identified by STARR-seq | journal = Science | volume = 339 | issue = 6123 | pages = 1074–7 | date = March 2013 | pmid = 23328393 | doi = 10.1126/science.1232542 | bibcode = 2013Sci...339.1074A | s2cid = 54488955 }}</ref> randomly sheared fragments of the ''Drosophila'' genome were placed downstream of a minimal promoter. Candidate enhancers amongst the randomly sheared fragments will transcribe themselves using the minimal promoter. By using sequencing as a readout and controlling for input amounts of each sequence the strength of putative enhancers are assayed by this method.

====Perturb-seq====
{{Main|Perturb-seq}}
[[File:Overview of Perturb-seq workflow.jpeg|thumb|517x517px|Overview of Perturb-seq workflow]]

Perturb-seq couples CRISPR mediated gene knockdowns with single-cell gene expression. Linear models are used to calculate the effect of the knockdown of a single gene on the expression of multiple genes.

===At the protein level===

====Yeast two-hybrid system====
{{Main|Two-hybrid screening}}
A yeast [[two-hybrid screening]] (Y2H) tests a "bait" protein against many potential interacting proteins ("prey") to identify physical protein–protein interactions. This system is based on a transcription factor, originally GAL4,<ref name=pmid2547163>{{cite journal | vauthors = Fields S, Song O | title = A novel genetic system to detect protein-protein interactions | journal = Nature | volume = 340 | issue = 6230 | pages = 245–6 | date = July 1989 | pmid = 2547163 | doi = 10.1038/340245a0 | bibcode = 1989Natur.340..245F | s2cid = 4320733 }}</ref> whose separate DNA-binding and transcription activation domains are both required in order for the protein to cause transcription of a reporter gene. In a Y2H screen, the "bait" protein is fused to the binding domain of GAL4, and a library of potential "prey" (interacting) proteins is recombinantly expressed in a vector with the activation domain. In vivo interaction of bait and prey proteins in a yeast cell brings the activation and binding domains of GAL4 close enough together to result in expression of a [[reporter gene]]. It is also possible to systematically test a library of bait proteins against a library of prey proteins to identify all possible interactions in a cell.

====MS and AP/MS====
{{Main|Protein mass spectrometry|Affinity purification}}
[[Mass spectrometry]] (MS) can identify proteins and their relative levels, hence it can be used to study protein expression. When used in combination with [[affinity purification]], [[mass spectrometry]] (AP/MS) can be used to study protein complexes, that is, which proteins interact with one another in complexes and in which ratios. In order to purify protein complexes, usually a "bait" protein is tagged with a specific protein or peptide that can be used to pull out the complex from a complex mix. The purification is usually done using an antibody or a compound that binds to the fusion part. The proteins are then digested into short [[peptide]] fragments and mass spectrometry is used to identify the proteins based on the mass-to-charge ratios of those fragments.

====Deep mutational scanning====
In deep mutational scanning, every possible amino acid change in a given protein is first synthesized.<ref>{{cite journal |last1=Araya |first1=Carlos |last2=Fowler |first2=Douglas |title=Deep mutational scanning: assessing protein function on a massive scale |journal=Trends in Biotechnology |date=September 29, 2011 |volume=29 |issue=9 |pages=435–442 |doi=10.1016/j.tibtech.2011.04.003 |pmid=21561674|pmc=3159719 }}</ref> The activity of each of these protein variants is assayed in parallel using barcodes for each variant.<ref>{{cite journal | vauthors = Penn WD, McKee AG, Kuntz CP, Woods H, Nash V, Gruenhagen TC, Roushar FJ, Chandak M, Hemmerich C, Rusch DB, Meiler J, Schlebach JP| title = Probing biophysical sequence constraints within the transmembrane domains of rhodopsin by deep mutational scanning| journal = Sci Adv | volume =  6 | issue = 10 | pages = eaay7505| date = March 2020 | pmid = 32181350 | doi = 10.1126/sciadv.aay7505| pmc = 7056298 | bibcode = 2020SciA....6.7505P}}</ref> By comparing the activity to the wild-type protein, the effect of each mutation is identified. While it is possible to assay every possible single amino-acid change due to combinatorics two or more concurrent mutations are hard to test. Deep mutational scanning experiments have also been used to infer protein structure and protein-protein interactions.<ref>{{cite journal |last1=Rollins |first1=N.J. |last2=Brock |first2=K.P. |last3=Poelwijk |first3=F.J |last4=Marks |first4=Debora |title=Inferring protein 3D structure from deep mutation scans |journal=Nature Genetics |date=2019 |volume=51 |issue=7 |pages=1170–1176 |doi=10.1038/s41588-019-0432-9 |pmid=31209393 |pmc=7295002 }}</ref> Deep Mutational Scanning is an example of a multiplexed assays of variant effect (MAVEs), a family of methods that involve mutagenesis of a DNA-encoded protein or regulatory element followed by a multiplexed assay for some aspect of function. MAVEs enable the generation of ‘variant effect maps’ characterizing aspects of the function of every possible single nucleotide change in a gene or functional element of interest. <ref>{{cite journal |last1=Fowler |first1=DM |last2=Adams |first2=DJ |last3=Gloyn |first3=AL |last4=Starita |first4=Lea |title=An Atlas of Variant Effects to understand the genome at nucleotide resolution |journal=Genome Biology |date=2023 |volume=24 |issue=1 |page=147 |doi=10.1186/s13059-023-02986-x |doi-access=free |pmid=37394429|pmc=10316620 }}</ref>

===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.

===Functional annotations for genes===

====Genome annotation====
{{Main|Genome project#Genome annotation}}
Putative genes can be identified by scanning a genome for regions likely to encode proteins, based on characteristics such as long [[open reading frames]], transcriptional initiation sequences, and [[polyadenylation]] sites. A sequence identified as a putative gene must be confirmed by further evidence, such as similarity to cDNA or EST sequences from the same organism, similarity of the predicted protein sequence to known proteins, association with promoter sequences, or evidence that mutating the sequence produces an observable phenotype.

====Rosetta stone approach====
The Rosetta stone approach is a computational method for de-novo protein function prediction. It is based on the hypothesis that some proteins involved in a given physiological process may exist as two separate genes in one organism and as a single gene in another. Genomes are scanned for sequences that are independent in one organism and in a single open reading frame in another. If two genes have fused, it is predicted that they have similar biological functions that make such co-regulation advantageous.