Design of guideRNA-Oligos (gRNA) for CRISPR/Cas gen. editing
Materials
20 mM dNTPs
10x Taq/Pfu buffer
2 Primers 10 μM
DNA template, 100 – 200 ng/μL
DNA polymerases (Pfu, Taq)
10x Taq/Pfu buffer
2 Primers 10 μM
DNA template, 100 – 200 ng/μL
DNA polymerases (Pfu, Taq)
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.