Hello everyone
I want to know regarding off target mutation detection in a CRISPR experiment. I have checked my gene target site through BLAST to know either the same target is available on other chromosome of rice or not. Some bp match on other chromosomes but PAM sequence is not available in those matched sequences. I want to know does PAM sequence is necessary for sequence present on other chromosomes to check off target mutation or we can take without PAM?
Thanks a lot
Hi,
PAM: Point/ Percent Accepted Mutation is the substitution/ change/ mutation of a single amino acid in the primary structure of a protein with another single amino acid, which is accepted by the processes of natural selection. This definition does not include all point mutations in the DNA of an organism.
You can use BLOSUM62 (BLOcks SUbstitution Matrix) as it is better than PAM since PAM ignores the point of evolutionary changes in the protein sequences.
I hope you find my answer convincing.
Regards,
Sahar Qazi Thanks a lot for your valuable response I will definitely consider your suggestion.
Hi,
My understanding was you wanted to know if targets without PAM: protospacer adjacent motif should be considered for off target editing.
In general the PAM has to be included for CRISPR editing so typically off target editing is only analyzed for those with a PAM. However, alternative PAMs may have to be considered, depending on which Cas protein you use.
For Cas9:
" As expected, sgRNAs with an NGG PAM had the highest proportion of active sgRNAs, 91% (Fig. 5a). Some alternative PAM sequences led to notable but smaller rates of sgRNA activity: NAG (26%), NCG (11%), and NGA (7%). While alternative PAMs should be avoided to maximize on-target activity, they must be considered as potential off-target sites. "
- Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016:34(2):184-91
- Hsu PD, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31:827–832.
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