- Incorrect ratio of EnGen® Spy Cas9 Nickase to guide RNA and/or target site. For complete digestion we recommend a 10:10:1 or higher molar ratio of EnGen® Spy Cas9 Nickase : guide RNA : target site.
- Suboptimal sequence of the guide RNA. Verify the sequence and design of the guide RNA.
- Poor quality guide RNA. Verify the integrity of the guide RNA by gel electrophoresis.
- Use of a suboptimal buffer. Please use NEBuffer 3.1, which is included with the enzyme.