title: Self-Cloning CRISPR.
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. © 2016 by John Wiley & Sons, Inc.
authors.affiliation: Hubrecht Institute and UMC Utrecht, Utrecht, The Netherlands.|Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine at Utrecht University, Utrecht, The Netherlands.|Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts.
authors.forename: Mandana
authors.initials: M
authors.lastname: Arbab
authors.affiliation: Hubrecht Institute and UMC Utrecht, Utrecht, The Netherlands.|Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts.
authors.forename: Richard I
authors.initials: RI
authors.lastname: Sherwood
mesh_terms: D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D059766:DNA End-Joining Repair / Q000235:genetics; D000072669:Gene Editing; D055879:Gene Knock-In Techniques; D059765:Homologous Recombination / Q000235:genetics; D006801:Humans; D051379:Mice
keywords: CRISPR/Cas9; GFP transgenesis; embryonic stem cells; gene editing; homologous recombination; knock-in; knockout