- Incorrect ratio of dCas9 to guide RNA, and target site. For complete binding we recommend a 10:10:1 or higher molar ratio (EnGen® Spy dCas9 : 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.