I am using cas-offinder to find mismatches. I found 1000s of sequences that differ from my guide RNA with minimum 4 nucleotides. I was thinking to consider the guide RNA with max 3 bases difference but could not find any. I am not sure will the gRNA bind if the difference is more than 3 bases? should i just check or off targets with only sequences with 4 bases mismatch?
Could anyone suggest me how should i proceed with this?
Off-targets have been reported by guideRNAs having as high as six mismatches, the process is not fully understood. However, in most instances 1 or 2 mismatches is enough to prevent recognition. I designed 5 different guideRNAs each based on a gene of interest but with 2 mismatches, and none of the five guideRNAs can cut the gene of interest. When i introduce the target site (containing the 2 mismatches) into the genome, all five guideRNAs can cut these sites.
It matters where the mismatches are located, specifically the relative distance to the PAM site (where a mismatch in the guideRNA close to the PAM site is more ‘severe’, thus leading to no recognition). I have personally used CHOPCHOP to predict my guideRNAs, so far always happy and without causing off-targets in my filamentous fungi (even confirmed no off-targets after 5 rounds of crispr with whole-genome sequencing). Note that most of these experiments were done in a ku70 deletion background (impaired non-homologous end-joining DNA repair). See Article Efficient marker free CRISPR/Cas9 genome editing for functio...
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line