Hello everyone,
I plan to use the CRISPR-Cas9 system in Arabidopsis thaliana and have designed 1600 kb long homology arms starting at 105 and 75 bs approximately from the cut site, The distance is right?. I want to insert a fragment of about 5kb.
I have already read this article: http://blog.addgene.org/crispr-101-homology-directed-repair , but I would like you to tell me your experiences or data from some other source
PS: My Arabidopsis have the gene Ku70 Knockout
Thanks in advance.
Hello, 1600 bp homology arms are OK (at least what we use in mice and rats, total homology up to 3 kb has been shown to be optimal). However, I would not start so far away from the cut site/DSB of Cas9. The closer the better! With 100 bp away your recombination frequency might be up to 10 fold less efficient than if your are just 10 bp away. Max. 10-20 bp distance is usually recommended. Still, it can work, e.g. if you could combine this with a positive selection marker this should be no problem.
regards-
Kai
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