I'm going to do point mutation correction by using CRISPR Ribonucleoprotein method and ss-ODN. Guide-RNA design softwares from the internet gave me few possible guideRNAs. I'm planning to use two gRNAs. Which is the best gRNA that I should use? Is it okay to use both gRNAs from single arm (left or right)?
*I have attached the possible gRNAs. Thank a lot.
Hello,
You say you are using two gRNAs. Are you using a double nicking approach or a double-cutting Cas9?
Thanks for the reply. Yes, I will be using the double nicking approach.
Looking at your set of gRNAs from your list, 5&6 or 3&5 are the only ones which will give the appropriate 5' overhang which appears to be better for HDR to occur. 5&6 would appear to be the best choice because they are closer together and generate a smaller overhang, but not all guides work with equal efficiency. So, it may be benefitial to try both combinations and see which pair works the best.
For additional reading:
https://www.ncbi.nlm.nih.gov/pubmed/23992846
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3818127/
- Badges
- Science topic
- Similar topics
- Computer Science and Engineering
- Bioinformatics