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Germline mutation
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== Current therapies == Many Mendelian disorders stem from [[Dominance (genetics)|dominant]] point mutations within genes, including [[cystic fibrosis]], [[Thalassemia|beta-thalassemia]], [[Sickle-cell disease|sickle-cell anemia]], and [[TayβSachs disease]].<ref name=":11"/> By inducing a double stranded break in sequences surrounding the disease-causing point mutation, a dividing cell can use the non-mutated strand as a template to repair the newly broken DNA strand, getting rid of the disease-causing mutation.<ref name=":6" /> Many different genome editing techniques have been used for genome editing, and especially germline mutation editing in germ cells and developing zygotes; however, while these therapies have been extensively studied, their use in human germline editing is limited.<ref>{{cite web|url=https://www.geneticsandsociety.org/internal-content/about-human-germline-gene-editing|title=About Human Germline Gene Editing {{!}} Center for Genetics and Society|website=www.geneticsandsociety.org|access-date=2017-12-01}}</ref> === CRISPR/Cas9 editing === [[File:DNA Repair after CRISPR-Cas9 cut.svg|thumb|254x254px|The CRISPR editing system is able to target specific DNA sequences and, using a donor DNA template, can repair mutations within this gene.]] This editing system induces a double stranded break in the DNA, using a guide RNA and effector protein Cas9 to break the DNA backbones at specific target sequences.<ref name=":6" /> This system has shown a higher specificity than TALENs or ZFNs due to the Cas9 protein containing homologous (complementary) sequences to the sections of DNA surrounding the site to be cleaved.<ref name=":6">{{cite journal | vauthors = Sander JD, Joung JK | title = CRISPR-Cas systems for editing, regulating and targeting genomes | journal = Nature Biotechnology | volume = 32 | issue = 4 | pages = 347β55 | date = April 2014 | pmid = 24584096 | doi = 10.1038/nbt.2842 | pmc=4022601}}</ref> This broken strand can be repaired in 2 main ways: homologous directed repair (HDR) if a DNA strand is present to be used as a template (either homologous or donor), and if one is not, then the sequence will undergo [[non-homologous end joining]] (NHEJ).<ref name=":6" /> NHEJ often results in insertions or deletions within the gene of interest, due to the processing of the blunt strand ends, and is a way to study gene knockouts in a lab setting.<ref name=":5">{{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 = 84β87 | date = January 2014 | pmid = 24336571 | doi = 10.1126/science.1247005 | pmc=4089965| bibcode = 2014Sci...343...84S }}</ref> This method can be used to repair a point mutation by using the [[Chromatid|sister chromosome]] as a template, or by providing a double stranded DNA template with the [[CRISPR]]/Cas9 machinery to be used as the repair template.<ref name=":6" /> This method has been used in both human and animal models (''[[Drosophila]]'', ''[[House mouse|Mus musculus]]'', ''and [[Arabidopsis]]''), and current research is being focused on making this system more specific to minimize off-target cleavage sites.<ref>{{cite journal | vauthors = Smith C, Gore A, Yan W, Abalde-Atristain L, Li Z, He C, Wang Y, Brodsky RA, Zhang K, Cheng L, Ye Z | title = Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs | language = en | journal = Cell Stem Cell | volume = 15 | issue = 1 | pages = 12β3 | date = July 2014 | pmid = 24996165 | doi = 10.1016/j.stem.2014.06.011 | pmc=4338993}}</ref> === TALEN editing === The [[Transcription activator-like effector nuclease|TALEN]] (transcription activator-like effector nucleases) genome editing system is used to induce a double-stranded DNA break at a specific locus in the genome, which can then be used to mutate or repair the DNA sequence.<ref name=":7">{{cite journal | vauthors = Bedell VM, Wang Y, Campbell JM, Poshusta TL, Starker CG, Krug RG, Tan W, Penheiter SG, Ma AC, Leung AY, Fahrenkrug SC, Carlson DF, Voytas DF, Clark KJ, Essner JJ, Ekker SC | title = In vivo genome editing using a high-efficiency TALEN system | journal = Nature | volume = 491 | issue = 7422 | pages = 114β8 | date = November 2012 | pmid = 23000899 | doi = 10.1038/nature11537 | pmc=3491146| bibcode = 2012Natur.491..114B }}</ref> It functions by using a specific repeated sequence of an amino acid that is 33-34 amino acids in length.<ref name=":7" /> The specificity of the DNA binding site is determined by the specific amino acids at positions 12 and 13 (also called the Repeat Variable Diresidue (RVD)) of this tandem repeat, with some RVDs showing a higher specificity for specific amino acids over others.<ref>{{cite journal | vauthors = Nemudryi AA, Valetdinova KR, Medvedev SP, Zakian SM | title = TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery | journal = Acta Naturae | volume = 6 | issue = 3 | pages = 19β40 | date = July 2014 | pmid = 25349712 | pmc = 4207558 | doi = 10.32607/20758251-2014-6-3-19-40 }}</ref> Once the DNA break is initiated, the ends can either be joined with NHEJ that induces mutations, or by HDR that can fix mutations.<ref name=":6" /> === ZFN editing === Similar to TALENs, [[zinc finger nuclease]]s (ZFNs) are used to create a double stranded break in the DNA at a specific locus in the genome.<ref name=":7" /> The ZFN editing complex consists of a [[Zinc finger|zinc finger protein]] (ZFP) and a restriction enzyme cleavage domain.<ref name=":8">{{cite journal | vauthors = Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD | title = Genome editing with engineered zinc finger nucleases | journal = Nature Reviews Genetics | volume = 11 | issue = 9 | pages = 636β46 | date = September 2010 | pmid = 20717154 | doi = 10.1038/nrg2842 | s2cid = 205484701 }}</ref> The ZNP domain can be altered to change the DNA sequence that the [[restriction enzyme]] cuts, and this cleavage event initiates cellular repair processes, similar to that of CRISPR/Cas9 DNA editing.<ref name=":8" /> Compared to CRISPR/Cas9, the therapeutic applications of this technology are limited, due to the extensive engineering required to make each ZFN specific to the desired sequence.<ref name=":8" />
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