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Capítulos
- 00:04Título
- 01:45Positive and Negative Screening for Evolving Cas9
- 04:47Delivery of Cas9 as an RNP with Truncated sgRNA
- 06:17RNP Delivery
- 07:27Transfection of Plasmids Encoding Sniper-Cas9 and sgRNA
- 09:01Targeted Deep Sequencing for the Analysis of On-target and Potential Off-target Sites
- 09:29Results: Analysis of Sniper-screen, On-target and Off-target Activities
- 10:37Conclusion
Here, we present a protocol to optimize CRISPR-Cas9 to achieve a higher specificity without the loss of on-target activity. We use a directed evolution approach called Sniper-screen to find a mutant Cas9 with the desired characteristics. Sniper-Cas9 is compatible with truncated single-guide RNAs and delivery in a ribonucleoprotein format, well-known strategies for achieving higher specificities.
