CRISPR/Cas9-mediated gene editing is a powerful technique that allows you to create knock-in/out mutations in any gene and any cell. CRISPR/Cas9 system can be delivered into cells in many forms, including plasmids, lentivirus, adeno-associated virus, and ribonucleoprotein (RNP) complexes. The RNP system, consisting of Cas9 proteins and single-guide RNAs (sgRNA), are gaining more popularity due many advantages it has over the other methods.
sgRNAs are traditionally produced through in vitro transcription using phage RNA polymerase. These in vitro transcribed sgRNAs contain a 5’-triphosphate, which was thought to trigger immune response in many cell types. Kim et al. recently published their study in Genome Research, which showed that sgRNAs with 5’-triphosphate modifications produced through in vitro-transcription can indeed induce innate immune responses and lead to cytotoxicity in human and murine cells. However, chemically synthesized sgRNAs without the 5’-triphosphate modifications demonstrated much better editing efficiency in cells, thus supporting that chemically synthesized sgRNAs are the most ideal reagent for CRISPR genome editing up till now.
GenScript synthesizes both unmodified and modified sgRNAs for targeted genome editing. Modified sgRNAs with 2’-O-methyl and phosphorothioate modifications at the first three 5’ and 3’ terminal residues are recommended for better stability and editing efficiency.
