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dc.contributor.authorDuda, Katarzynaen_US
dc.contributor.authorLonowski, Lindsey A.en_US
dc.contributor.authorKofoed-Nielsen, Michaelen_US
dc.contributor.authorIbarra, Adrianaen_US
dc.contributor.authorDelay, Catherine M.en_US
dc.contributor.authorKang, Qiaohuaen_US
dc.contributor.authorYang, Zhangen_US
dc.contributor.authorPruett-Miller, Shondra M.en_US
dc.contributor.authorBennett, Eric P.en_US
dc.contributor.authorWandall, Hans H.en_US
dc.contributor.authorDavis, Gregory D.en_US
dc.contributor.authorHansen, Steen H.en_US
dc.contributor.authorFrödin, Mortenen_US
dc.date.accessioned2014-07-07T17:04:14Z
dc.date.issued2014en_US
dc.identifier.citationDuda, K., L. A. Lonowski, M. Kofoed-Nielsen, A. Ibarra, C. M. Delay, Q. Kang, Z. Yang, et al. 2014. “High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs.” Nucleic Acids Research 42 (10): e84. doi:10.1093/nar/gku251. http://dx.doi.org/10.1093/nar/gku251.en
dc.identifier.issn0305-1048en
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:12406795
dc.description.abstractTargeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing efficiencies by these tools. Briefly, 2A peptide-coupled co-expression of fluorescent protein and nuclease was combined with fluorescence-activated cell sorting (FACS) to allow for efficient isolation of cell populations with increasingly higher nuclease expression levels, which translated into increasingly higher genome editing rates. For ZFNs, this approach, combined with delivery of donors as single-stranded oligodeoxynucleotides and nucleases as messenger ribonucleic acid, enabled high knockin efficiencies in demanding applications, including biallelic codon conversion frequencies reaching 30–70% at high transfection efficiencies and ∼2% at low transfection efficiencies, simultaneous homozygous knockin mutation of two genes with ∼1.5% efficiency as well as generation of cell pools with almost complete codon conversion via three consecutive targeting and FACS events. Observed off-target effects were minimal, and when occurring, our data suggest that they may be counteracted by selecting intermediate nuclease levels where off-target mutagenesis is low, but on-target mutagenesis remains relatively high. The method was also applicable to the CRISPR/Cas9 system, including CRISPR/Cas9 mutant nickase pairs, which exhibit low off-target mutagenesis compared to wild-type Cas9.en
dc.language.isoen_USen
dc.publisherOxford University Pressen
dc.relation.isversionofdoi:10.1093/nar/gku251en
dc.relation.hasversionhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041425/pdf/en
dash.licenseLAAen_US
dc.titleHigh-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairsen
dc.typeJournal Articleen_US
dc.description.versionVersion of Recorden
dc.relation.journalNucleic Acids Researchen
dash.depositing.authorHansen, Steen H.en_US
dc.date.available2014-07-07T17:04:14Z
dc.identifier.doi10.1093/nar/gku251*
dash.authorsorderedfalse
dash.contributor.affiliatedHansen, Steen


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