I have a project concerning BRCA1 knockout,
by using crispr.mit.edu tool (V2), I cannot find a gRNA that matches perfectly my target sequence.
I'va selected the whole BRCA1 gene and selected the first 3 exons to do a cas9 assay.
The first 2 guides show a score of 99%, which is good but the sequence simply is not the same à the exon targeted sequence,
do you have an explanation for that ?
Despite the 99% score I'm not confortable with the fact that my guide won't be as same as the targeted genome sequence.
Here is the example :
CRISPR tool type cas9
guide sequence TCTGGCTGCACAACCACAAT
5' pam sequence (N/A)
3' pam sequence TGG
target exon name EXON_1
target exon start BRCA1 3348
on-target strand 1
Hsu, 2013 score 99.85011394611143
On-target cut site
__ __ __ __ __ __ __ __ __ __ __ __ AAGATTTATAAATAACCATCCTCTCCCTTGCCCTTCAACATTATAGCTAAACAAAAATAA TTATTTTTGTTTAGCTATAATGTTGAAGGGCAAGGGAGAGGATGGTTATTTATAAATCTT __ __
On-target exon sequence
EXON_1 (hg38 BRCA1) guide TCTGGCTGCACAACCACAATTGG
|NC_000021.9 38519815 +||14:T>C,16:G>C,18:T>A|
|NC_000011.10 98392516 +||6:A>T,7:A>G,8:A>C|
|NC_000016.10 87203142 +||4:A>G,15:C>A,19:A>T|