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

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