Design of guideRNA-Oligos (gRNA) for CRISPR/Cas gen. editing
Materials
The genomic sequence (with exon/intron structure) of your target gene
Computer with internet-access to the Chop-Chop website (https://chopchop.rc.fas.harvard.edu/)
gRNA Oligos (sense/antisense) will be cloned in one of several vectors harboring a U6-promoter-driven RNA expression cassette
Computer with internet-access to the Chop-Chop website (https://chopchop.rc.fas.harvard.edu/)
gRNA Oligos (sense/antisense) will be cloned in one of several vectors harboring a U6-promoter-driven RNA expression cassette
Vectors used are: pBluescript_U6-MCS (Plasmid #3468) pBluescript_U6-MCS gRNA_Cerulean (Plasmid #xxx) pX45xxx pLenti
Design principles for 20 bp gRNA
- Use the Chop-Chop website to find an appropriate gRNA target site upstream of a PAM sequence with no or low off-target probability
- In general, try to target the first coding exon downstream of the initiating ATG start codon. Check that there is not an alternative ATG start codon in the proper reading frame nearby
- Make sure to use the correct genomic sequence (correct species) and target gene
- in case of multiple splice variants, try to target a common exon
Fig. 1: Exemplary scheme for the design of gRNA.