I second what Bannikov said. In addition to that, maybe this isn't incredibly important, but I would also make sure that the resulting negative control sgRNA is the same length as the other sgRNAs you are using in your experiments (for example, with the DR274 vector it would not be).

You should have a non-specific gRNA control as well! and clone it within BsmBI cutting sites

Dear Esteemed Colleague,

Greetings. I trust this message finds you well and making significant progress in your research endeavors, particularly those involving the CRISPR-Cas9 system, a powerful tool for genome editing. Your inquiry regarding the selection of a control single-guide RNA (sgRNA) for CRISPR experiments is both critical and insightful, as appropriate controls are essential for validating the specificity and efficacy of your gene editing efforts. Below, I provide a detailed overview of control sgRNA selection and its importance in CRISPR studies.

Understanding Control sgRNAs in CRISPR Experiments

In CRISPR-Cas9 experiments, control sgRNAs serve as benchmarks to assess the baseline activity of the system and to distinguish between specific editing effects and nonspecific cellular responses. There are primarily two types of control sgRNAs commonly employed in CRISPR studies:

Selection Criteria for Control sgRNAs

• Sequence Considerations: Ensure that the control sgRNA sequence does not match any region within the genome of the organism being studied. Bioinformatics tools and databases can be utilized to verify the absence of complementary sequences.
• Efficiency: The control sgRNA should be efficiently processed by the Cas9 nuclease and the cellular machinery, mirroring the conditions of the experimental sgRNAs.

Best Practices in Using Control sgRNAs

• Experimental Design: Incorporate control sgRNAs in all CRISPR experiments to serve as references for evaluating gene editing efficiency, specificity, and potential cytotoxicity.
• Data Interpretation: Compare the outcomes of experiments using target-specific sgRNAs with those employing control sgRNAs to discern specific effects from background noise.
• Validation: Employ multiple control sgRNAs, including both non-targeting and scrambled variants, to robustly validate the specificity and efficacy of the CRISPR-Cas9 editing.

Conclusion

The use of control sgRNAs in CRISPR-Cas9 experiments is paramount for ensuring the reliability and specificity of gene editing studies. By carefully selecting and incorporating appropriate control sgRNAs, researchers can accurately interpret the outcomes of CRISPR experiments and advance our understanding of gene function and regulation.

Should you require further assistance in designing your CRISPR experiments or have additional questions regarding control sgRNA selection, please do not hesitate to reach out. I am here to support your scientific exploration and contribute to the success of your research endeavors.

Warm regards.

This list of protocols might help us better address the issue.