Lentiviral CRISPR Targeting DNMT3A

The cDNA sequence encoding sgRNA which targets a conserved sequence in exon 7 of human DNMT3A gene was synthesized and subcloned into LentiCRISPR-v2 plasmid (Addgene 52961, kindly provided by Dr. Jian Huang at Temple University, Philadelphia, PA) to make the lentiDNMT3A-sgRNA-Cas9 construct. Briefly, the forward and reverse primers including 20 bp target DNMT3A sequence and BsmbI sticky ends were annealed and inserted into the LentiCRISPR-v2 plasmid digested with FastDigest Esp3I (Thermo Fisher Scientific, #FD0454) (Fig. 1b). sgRNA primer sequences have been reported by Gundry MC et al. previously and were shown in Table 1 [22 (link)].Fig. 1

Schematic diagram of sgRNA targeting DNMT3A. a The structure of DNMT3A gene and the three common transcripts. Black vertical lines: exons. Horizontal lines: introns. Arrow: the location of sgRNA targeting exon 7. b The structure of lentiCRISPR v2 plasmid. The arrows indicate the sgRNA sequence