Editing Gene knockout (section)

===Site-specific nucleases===
[[File:Frameshift mutations (13080927393).jpg|thumb|303x303px|Frameshift mutation resulting from a single base pair deletion, causing altered amino acid sequence and premature stop codon]]There are currently three methods in use that involve precisely targeting a DNA sequence in order to introduce a double-stranded break. Once this occurs, the cell's repair mechanisms will attempt to repair this double stranded break, often through [[non-homologous end joining]] (NHEJ), which involves directly ligating the two cut ends together.<ref name=":1">{{Cite journal|last1=Santiago|first1=Yolanda|last2=Chan|first2=Edmond|last3=Liu|first3=Pei-Qi|last4=Orlando|first4=Salvatore|last5=Zhang|first5=Lin|last6=Urnov|first6=Fyodor D.|last7=Holmes|first7=Michael C.|last8=Guschin|first8=Dmitry|last9=Waite|first9=Adam|date=2008-04-15|title=Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases|journal=Proceedings of the National Academy of Sciences|volume=105|issue=15|pages=5809–5814|doi=10.1073/pnas.0800940105|issn=0027-8424|pmid=18359850|pmc=2299223|doi-access=free}}</ref> This may be done imperfectly, therefore sometimes causing insertions or deletions of base pairs, which cause [[frameshift mutation]]s. These mutations can render the gene in which they occur nonfunctional, thus creating a knockout of that gene. This process is more efficient than homologous recombination, and therefore can be more easily used to create biallelic knockouts.<ref name=":1" />

====Zinc-fingers====
{{Main|Zinc-finger nuclease}}
Zinc-finger nucleases consist of DNA binding domains that can precisely target a DNA sequence.<ref name=":1" /> Each zinc-finger can recognize codons of a desired DNA sequence, and therefore can be modularly assembled to bind to a particular sequence.<ref name=":2" /> These binding domains are coupled with a [[Restriction enzyme|restriction endonuclease]] that can cause a double stranded break (DSB) in the DNA.<ref name=":1" /> Repair processes may introduce mutations that destroy functionality of the gene.{{cn|date=December 2023}}

====TALENS====
{{Main|Transcription activator-like effector nuclease}}
Transcription activator-like effector nucleases ([[TALENs]]) also contain a DNA binding domain and a nuclease that can cleave DNA.<ref name=":3">{{Cite journal|last1=Joung|first1=J. Keith|last2=Sander|first2=Jeffry D.|date=January 2013|title=TALENs: a widely applicable technology for targeted genome editing|journal=Nature Reviews Molecular Cell Biology|volume=14|issue=1|pages=49–55|doi=10.1038/nrm3486|pmid=23169466|issn=1471-0080|pmc=3547402}}</ref> The DNA binding region consists of amino acid repeats that each recognize a single base pair of the desired targeted DNA sequence.<ref name=":2">{{Cite journal|last1=Gaj|first1=Thomas|last2=Gersbach|first2=Charles A.|last3=Barbas|first3=Carlos F.|title=ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering|journal=Trends in Biotechnology|volume=31|issue=7|pages=397–405|doi=10.1016/j.tibtech.2013.04.004|pmid=23664777|pmc=3694601|year=2013}}</ref> If this cleavage is targeted to a gene coding region, and NHEJ-mediated repair introduces insertions and deletions, a frameshift mutation often results, thus disrupting function of the gene.<ref name=":3" />

====CRISPR/Cas9====
{{Main|CRISPR}}
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a genetic engineering technique that allows for precise editing of the genome. One application of CRISPR is gene knockout, which involves disabling or "knocking out" a specific gene in an organism.{{cn|date=December 2023}}

The process of gene knockout with CRISPR involves three main steps: designing a guide RNA (gRNA) that targets a specific location in the genome, delivering the gRNA and a Cas9 enzyme (which acts as a molecular scissors) to the target cell, and then allowing the cell to repair the cut in the DNA. When the cell repairs the cut, it can either join the cut ends back together, resulting in a non-functional gene, or introduce a mutation that disrupts the gene's function.

This technique can be used in a variety of organisms, including bacteria, yeast, plants, and animals, and it allows scientists to study the function of specific genes by observing the effects of their absence. CRISPR-based gene knockout is a powerful tool for understanding the genetic basis of disease and for developing new therapies. 

It is important to note that CRISPR-based gene knockout, like any genetic engineering technique, has the potential to produce unintended or harmful effects on the organism, so it should be used with caution.<ref name=":2" /><ref>{{Cite journal|last1=Ni|first1=Wei|last2=Qiao|first2=Jun|last3=Hu|first3=Shengwei|last4=Zhao|first4=Xinxia|last5=Regouski|first5=Misha|last6=Yang|first6=Min|last7=Polejaeva|first7=Irina A.|last8=Chen|first8=Chuangfu|date=2014-09-04|title=Efficient Gene Knockout in Goats Using CRISPR/Cas9 System|journal=PLOS ONE|volume=9|issue=9|pages=e106718|doi=10.1371/journal.pone.0106718|pmid=25188313|pmc=4154755|bibcode=2014PLoSO...9j6718N|issn=1932-6203|doi-access=free}}</ref> The coupled Cas9 will cause a double stranded break in the DNA.<ref name=":2" /> Following the same principle as zinc-fingers and TALENs, the attempts to repair these double stranded breaks often result in frameshift mutations that result in an nonfunctional gene.<ref name=":2" /> Non invasive CRISPR-Cas9 technology has successfully knocked out a gene associated in depression and anxiety in mice, being the first successful delivery passing through the [[blood–brain barrier]] to enable gene modification.<ref>{{Cite web |date=2023-06-21 |title=First-of-its-kind noninvasive CRISPR method knocks out anxiety gene |url=https://newatlas.com/medical/intranasal-crispr-gene-editing-reduces-anxiety-in-mice/ |access-date=2024-01-18 |website=New Atlas |language=en-US}}</ref>