Use of CRISPR-Cas9 has revolutionized targeted genome editing. The process requires the Cas9 enzyme and guide RNA (gRNA), which directs Cas9 to the target DNA location. The gRNA can be generated in various forms using different methods: gRNA can be expressed from a plasmid as a single guide RNA (sgRNA), transcribed in vitro as an sgRNA (IVT sgRNAs), or chemically synthesized as an sgRNA or two-part complex (crRNA duplexed with tracrRNA). Chemically synthesized gRNAs can be modified to enhance editing efficiency by stabilizing the gRNA against nuclease degradation, and also by reducing the risk of an innate immune response often seen with IVT sgRNAs.
In this webinar, Ashley Jacobi compares the on-target and off-target editing events observed with different gRNA formats in transformed and primary cell lines. She provides guidelines on when to use each gRNA in the context of your Cas9 source (plasmid, mRNA, ribonucleoprotein complex) and cell type.