Self‐Cloning CRISPR

AbstractCRISPR/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.