NDLI: Self‐Cloning CRISPR

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Self‐Cloning CRISPR

Content Provider	Scilit
Author	Arbab, Mandana Sherwood, Richard I.
Copyright Year	2016
Description	Journal: Current Protocols in Stem Cell Biology CRISPR/Cas9‐gene editing has emerged as a revolutionary technology to easily modify specific genomic loci by designing complementary sgRNA sequences and introducing these into cells along with Cas9. Self‐cloning CRISPR/Cas9 (scCRISPR) uses a self‐cleaving palindromic sgRNA plasmid (sgPal) that recombines with short PCR‐amplified site‐specific sgRNA sequences within the target cell by homologous recombination to circumvent the process of sgRNA plasmid construction. Through this mechanism, scCRISPR enables gene editing within 2 hr once sgRNA oligos are available, with high efficiency equivalent to conventional sgRNA targeting: >90% gene knockout in both mouse and human embryonic stem cells and cancer cell lines. Furthermore, using PCR‐based addition of short homology arms, we achieve efficient site‐specific knock‐in of transgenes such as GFP without traditional plasmid cloning or genome‐integrated selection cassette (2% to 4% knock‐in rate). The methods in this paper describe the most rapid and efficient means of CRISPR gene editing. © 2016 by John Wiley & Sons, Inc.
Related Links	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4993524/pdf
ISSN	19417322
e-ISSN	19388969
DOI	10.1002/cpsc.14
Journal	Current Protocols in Stem Cell Biology
Issue Number	1
Volume Number	38
Language	English
Publisher	Wiley-Blackwell
Publisher Date	2016-08-17
Access Restriction	Open
Subject Keyword	Journal: Current Protocols in Stem Cell Biology Biotechnology and Microbiology Embryonic Stem Cells Homologous Recombination
Content Type	Text
Subject	Cell Biology Developmental Biology Medicine

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