Abstract
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. (c) 2016 by John Wiley & Sons, Inc.
| Original language | English |
|---|---|
| Pages (from-to) | 5b.5.1-5b.5.16 |
| Journal | Current Protocols in Stem Cell Biology |
| Volume | 38 |
| DOIs | |
| Publication status | E-pub ahead of print - 17 Aug 2016 |
Keywords
- CRISPR/Cas9 GFP transgenesis embryonic stem cells gene editing homologous recombination knock-in knockout