Self-Cloning CRISPR

Curr Protoc Stem Cell Biol. 2016 Aug 17:38:5B.5.1-5B.5.16. doi: 10.1002/cpsc.14.


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.

Keywords: CRISPR/Cas9; GFP transgenesis; embryonic stem cells; gene editing; homologous recombination; knock-in; knockout.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cloning, Molecular / methods*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • DNA End-Joining Repair / genetics
  • Gene Editing
  • Gene Knock-In Techniques
  • Homologous Recombination / genetics
  • Humans
  • Mice