This protocol details the complete CRISPR-Cas9 gene editing workflow in mammalian cells (HEK293T): gRNA design, plasmid construction, cell transfection, T7E1 cleavage assay for editing efficiency, and Sanger sequencing validation.
Use CRISPOR or Benchling to enter the target gene sequence and select candidate gRNAs with high on-target score.
Anneal forward and reverse oligos into double-stranded inserts; digest backbone with BbsI; ligate and transform.
HEK293T cells at 70 to 80 percent confluence; transfect the Cas9-gRNA plasmid with Lipofectamine 3000.
Extract genomic DNA, PCR-amplify the target locus, digest with T7E1 endonuclease, and quantify Indel frequency on an agarose gel.
Send PCR products for Sanger sequencing; analyze with the TIDE tool to quantify Indels and validate edits.
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