CRISPR-mediated gene editing in cells

Guide RNA (gRNA) was designed with http://crispr.mit.edu. The gRNAs were cloned into the pCAG-mCherry-gRNA vector (Addgene #87110). For the expression of Cas9 and GFP (Cas9-2A-GFP), the pCAG-1BPNLS-Cas9-1BPNLS-2AGFP plasmid (Addgene #87109) was used (Suzuki et al., 2016 (link)). The sequences for the gRNA target and ssODN used to repair mutant alleles are as follows: Exon 4-gRNA: GGATCACGCCAGTCTGGAGTAGG. ERCC6-ssODN, 5′-CTAAAGAGACACCCTCCACTGACTACAGGCATCAGGCATCAATTCAAGAACACAGAGAAACTGCTCCTAGCATCCTCACCTGCATCCTCtTCCAGACTGGCGTGATCTAGTTCAATTTTCACCTCTG-3′.

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Wang S., Min Z., Ji Q., Geng L., Su Y., Liu Z., Hu H., Wang L., Zhang W., Suzuiki K., Huang Y., Zhang P., Tang T.S., Qu J., Yu Y., Liu G.H, & Qiao J. (2019). Rescue of premature aging defects in Cockayne syndrome stem cells by CRISPR/Cas9-mediated gene correction. Protein & Cell, 11(1), 1-22.

Publication 2019

pCAG-mCherry-gRNA vector

Alleles Crispr Ercc6 Exon Plasmid Rna sequences Vector

Corresponding Organization : Chinese Institute for Brain Research

Other organizations : Beijing Institute of Genomics, Osaka University

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Variable analysis

independent variables

Guide RNA (gRNA) designed using http://crispr.mit.edu
Cloning of gRNAs into the pCAG-mCherry-gRNA vector (Addgene #87110)
Expression of Cas9 and GFP (Cas9-2A-GFP) using the pCAG-1BPNLS-Cas9-1BPNLS-2AGFP plasmid (Addgene #87109)

dependent variables

Repair of mutant alleles using the provided ssODN sequence

control variables

Not explicitly mentioned

controls

No positive or negative controls explicitly mentioned

Annotations

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