Show simple item record

dc.contributor.authorLi, Kaien_US
dc.contributor.authorWang, Gangen_US
dc.contributor.authorAndersen, Troelsen_US
dc.contributor.authorZhou, Pingzhuen_US
dc.contributor.authorPu, William T.en_US
dc.date.accessioned2014-09-08T15:37:24Z
dc.date.issued2014en_US
dc.identifier.citationLi, Kai, Gang Wang, Troels Andersen, Pingzhu Zhou, and William T. Pu. 2014. “Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System.” PLoS ONE 9 (8): e105779. doi:10.1371/journal.pone.0105779. http://dx.doi.org/10.1371/journal.pone.0105779.en
dc.identifier.issn1932-6203en
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:12785935
dc.description.abstractDesigner nucleases such as TALENS and Cas9 have opened new opportunities to scarlessly edit the mammalian genome. Here we explored several parameters that influence Cas9-mediated scarless genome editing efficiency in murine embryonic stem cells. Optimization of transfection conditions and enriching for transfected cells are critical for efficiently recovering modified clones. Paired gRNAs and wild-type Cas9 efficiently create programmed deletions, which facilitate identification of targeted clones, while paired gRNAs and the Cas9D10A nickase generated smaller targeted indels with lower chance of off-target mutagenesis. Genome editing is also useful for programmed introduction of exogenous DNA sequences at a target locus. Increasing the length of the homology arms of the homology-directed repair template strongly enhanced targeting efficiency, while increasing the length of the DNA insert reduced it. Together our data provide guidance on optimal design of scarless gene knockout, modification, or knock-in experiments using Cas9 nuclease.en
dc.language.isoen_USen
dc.publisherPublic Library of Scienceen
dc.relation.isversionofdoi:10.1371/journal.pone.0105779en
dc.relation.hasversionhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4148324/pdf/en
dash.licenseLAAen_US
dc.subjectBiology and Life Sciencesen
dc.subjectAgricultureen
dc.subjectAgricultural Biotechnologyen
dc.subjectGenetically Modified Organismsen
dc.subjectBiotechnologyen
dc.subjectGenetic Engineeringen
dc.subjectTransgenic Engineeringen
dc.subjectGeneticsen
dc.subjectGene Disruptionen
dc.subjectGene Functionen
dc.subjectGene Fusionen
dc.titleOptimization of Genome Engineering Approaches with the CRISPR/Cas9 Systemen
dc.typeJournal Articleen_US
dc.description.versionVersion of Recorden
dc.relation.journalPLoS ONEen
dash.depositing.authorPu, William T.en_US
dc.date.available2014-09-08T15:37:24Z
dc.identifier.doi10.1371/journal.pone.0105779*
dash.contributor.affiliatedPu, William


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record