I am designing CRISPR Cas 9 guide strands to an intronic sequence for knock-in experiments.
I am checking for SNPs/variants in the locations of these guides using the NCBI portal of databases, specifying the chromosomal range of interest.
In doing so, the search output gives me no SNP result for this particular intronic sequence. This is great (less work for me!), but is it real?
I have done this previously with another gene's intronic sequence and the output was about 1200 pages of of reported SNPs and variants, which is why I am a bit unsure if this result is normal.
Any help would be appreciated. Thank you.
Dear Elena,
This can vary a lot. One reason could be that are much less genetic studieso be on the safe side, I would re-check at Ensembl.org. Choose "show variants" under "configure this page".
Best, Uwe
This is extraordinary that the intron has no SNPs, even in coding regions one finds SNPs (neutral), so there must be a reason based on sequence/structural conservation of the intron. Could be that the intron undergoes alternative splicing and the intron is retained in the coding mRNA -which has an essential function?
I guess you have identified some guide K-mer(s) gRNA sequences of around 20bp which are unique within your targeted genome, furthermore these sequences are located within your annotated intron and have a PAM 3' to your sequence. So already you have significant constraints in terms of sequence conservation, so I would not be surprised if there are no annotated SNPs within your quide 20bp K-mer.
To gain confidence in reducing the off-targets, you may want to impose further constraints on the uniqueness of the guide K-mer noting that it is the first 10bp (adjacent to the PAM) which must exactly match, and when binding extends 3' -> 5' then a couple of mismatches may not stop binding extension. If you insist that the gRNA K-mer is at least 2 substitutions (Hammings) away from any other same sized K-mer in the genome then you would greatly reduce off-targets.
WRT to Andy Pereira's comment - many introns contain ncRNAs which can be under more evolutionary constraint than coding Exons. These ncRNAs may/not be annotated, and can be rather long - 200bp or more. For extreme examples then 'google' for the 'Ultraconserved Elements' paper, no explanation to date for their existence other than a few instances of elements identified as containing ncRNAs.
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