Self-Cloning CRISPR

M. Arbab, R. I. Sherwood

Research output: Contribution to journal/periodicalArticleScientificpeer-review

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 languageEnglish
Pages (from-to)5b.5.1-5b.5.16
JournalCurrent Protocols in Stem Cell Biology
Volume38
DOIs
Publication statusE-pub ahead of print - 17 Aug 2016

Keywords

  • CRISPR/Cas9 GFP transgenesis embryonic stem cells gene editing homologous recombination knock-in knockout

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