Pcag mcherry grna vector

CRISPR/Cas9-mediated gene knockout was performed as previously described (44 (link),45 (link)). Briefly, guide RNA was designed to target the ATG codon within exon 3 of the ZKSCAN3 gene, and then cloned into pCAG-mCherry-gRNA vector (Addgene #87110). H9 hESCs were cultured on Matrigel-coated plates and treated with ROCK inhibitor Y-27632 (Selleck) for 24 hr before being electroporated by 4D-Nucleofector (Lonza) with both 7 μg gRNA vector and 14 μg pCAG-1BPNLS-Cas9-1BPNLS-2AGFP (Addgene #87109). Subsequently, cells were plated on Matrigel-coated plates in mTeSR medium for 48 hr. GFP/mCherry double-positive cells were then purified by fluorescence activated cell sorting (FACS) and cultured on MEF feeders in hESC culture medium. Guide RNA sequences for targeting ZKSCAN3 locus and primers used for gene targeting and off-target identification are listed in Supplementary Table S1.

CRISPR/Cas9-mediated BMAL1 gene editing in hESCs was performed following previously described protocols (56–58 ). Briefly, a guide RNA targeting exon 8 of BMAL1 was cloned into a pCAGmCherry-gRNA vector (#87110, Addgene), marking the plasmid as BMAL1-gRNA-mCherry. Next, BMAL1^+/+ hESCs were resuspended in the plasmid cocktail that contained BMAL1-gRNA-mCherry and pCAG-1BPNLS-Cas9-1BPNLS-2AGFP plasmid (#87109, Addgene) in Opti-MEM (Thermo Fisher Scientific). After electroporation by a 4D-Nucleofector system (Lonza), hESCs were seeded on Matrigel precoated plates in mTeSR containing ROCK inhibitor (Y-27632, TOCRIS). Then, mCherry and GFP dual-positive cells were sorted by fluorescence-activated cell sorting (FACSAria IIIu, BD) and seeded for further expansion. hESC clones were then genotyped by genomic PCR and sequencing. The introduced single nucleic acid base in the exon 8 of BMAL1 resulted in a frameshift mutation and premature termination of translation. Successful knockout of BMAL1 in hESCs was further verified by western blot detection. Sequences of the sgRNA used for gene editing and primers used for genotyping were listed in Supplementary Table S1.