FAQ: How do I design a single guide RNA for use with EnGen SpRY Cas9?

EnGen SpRY Cas9 utilizes the same single guide RNA scaffold as other S. pyogenes Cas9s:


  • 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:

M0669 sgRNA Options

For every desired double stranded break on the DNA target, two single guide RNAs are available (targeting either strand of the DNA) when using EnGen SpRY Cas9.