Sherwood, Richard I.
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CitationArbab, Mandana, Sharanya Srinivasan, Tatsunori Hashimoto, Niels Geijsen, and Richard I. Sherwood. 2015. “Cloning-free CRISPR.” Stem Cell Reports 5 (5): 908-917. doi:10.1016/j.stemcr.2015.09.022. http://dx.doi.org/10.1016/j.stemcr.2015.09.022.
AbstractSummary We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:23993512
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