Generating a New sgRNA Vector, pGL3-U6-sgRNA-PGK-mRFP-T2A-PuroR, to Improve Base Editing

CRISPR/Cas9-mediated base editing introduces point mutations in cellular DNA by exploiting target-specific single guide RNA (sgRNA) along with a genetically modified Cas9. Existing plasmid vectors for sgRNA expression contain either fluorescent marker or an antibiotic resistance cassette but not both, preventing simultaneous monitoring and enrichment of transfected host cells. In this study, we introduced into pGL3-U6-sgRNA-PGK-puromycin, popular vector available at Addgene. Specifically, the cDNAs mRFP T2A linker were inserted between hPGK promoter puromycin gene (PuroR). After correct insertion was verified sequencing, new plasmid, pGL3-U6-sgRNA-PGK-mRFP-T2A-PuroR, utilized to generate stop codon second exon Munc13-1 RBL-2H3 Both functioned accordingly process. This therefore represents useful addition CRISPR tool kit.