I want to add a tag to the 3' end of my target gene using Crispr-Cas9 system. What can I do if the appropriate sgRNA is far from the 3' end of the protein?
Hello, Nowadays, there are a lot of knows Cas9 type (S. pyogenes, S.aureus, N. meningitidis,...) and CpfI proteins. They all recognize a different PAM motif. So maybe you can find a good Cas9 protein for making DSB at the right position, depending on your sequence.
And, for adding your tag, you can depending on the size of your tag:
Making a HR donor with long homology arms, classical way.
Using modified oligos with short homologies on the extremities of the oligo (perfect strategy for a short tag, like 3xflag, HA,...). You will find some explanations on this publication:
http://www.cell.com/cell-reports/fulltext/S2211-1247%2816%2930104-8
Here is a link that we built, to help finding your PAM motifs, with different Cas9 types, in several genomes.
http://crispor.tefor.net/crispor.py
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