EnGen SpRY Cas9 utilizes the same single guide RNA scaffold as other S. pyogenes Cas9s:
5´-NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3´
- N’s represent the 20 nucleotide sequence specific to the DNA target
- A synthetic single guide RNA may be purchased from an oligonucleotide synthesis company
- Alternatively, if higher yields are required, the NEB EnGen sgRNA Synthesis Kit, S. pyogenes (NEB #E3322) may be purchased
The following figure illustrates how to design guide(s) for a specific DNA cut site:
