Self-Cloning CRISPR

TY - JOUR

T1 - Self-Cloning CRISPR

AU - Arbab, M.

AU - Sherwood, R. I.

N1 - 1938-8969 Arbab, Mandana Sherwood, Richard I RL1 DE019021/DE/NIDCR NIH HHS/United States K01 DK101684/DK/NIDDK NIH HHS/United States U01 HG007037/HG/NHGRI NIH HHS/United States P01 NS055923/NS/NINDS NIH HHS/United States R01 HG008363/HG/NHGRI NIH HHS/United States Journal Article United States Curr Protoc Stem Cell Biol. 2016 Aug 17;38:5B.5.1-5B.5.16. doi: 10.1002/cpsc.14.

PY - 2016/8/17

Y1 - 2016/8/17

N2 - 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.

AB - 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.

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

U2 - 10.1002/cpsc.14

DO - 10.1002/cpsc.14

M3 - Article

SN - 1938-8969

VL - 38

SP - 5b.5.1-5b.5.16

JO - Current Protocols in Stem Cell Biology

JF - Current Protocols in Stem Cell Biology

ER -