Synthesis of 100 nt long sgRNAs was traditionally possible through in vitro-transcription (IVT) using phage RNA polymerase. These in vitro transcribed sgRNAs contain a 5’-triphosphate, which was thought to trigger immune response in many cell types. A recent study 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 now3.
3. Kim et al. CRISPR RNAs trigger innate immune responses in human cells. Genome Res. 2018. 28: 367-373.