Self-Cloning CRISPR.

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 palindromicsgRNA plasmid (sgPal) that recombines with short PCR-amplified site-specific sgRNA sequences within the target cell by homologous recombinationto 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 knockoutin 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 CRISPRgene editing. (c) 2016 by John Wiley & Sons, Inc.