I would like to knockout a specific gene in the developing brain by Crisp-Cas9 and in utero electroporation. In parallel, I need to overexpress - by coelectroporating plasmids- the mutant forms to assess their differential phenotypes following endogenous gene knockout.
How to avoid that mutant plasmids are silenced by the same Crisp-Cas9 I need to knockout the endogenous gene?
You can introduce silent mutations in the region targeted by your gRNA and/or the PAM sequence. This way your construct will not be targeted.
Thank you Yoav,
I followed the same approach for sh-RNA silencing and it worked well. But I have no experience with Crisp-Cas9 gene editing yet..I will try in this way
Maybe helpful for you. https://www.researchgate.net/post/How_to_put_back_a_gene_when_using_Lenticrispr_KO
You could do direct site mutagenesis on the gRNA target on the plasmid, to break the PAM. Make sure that the new nucleotide that you insert does not introduce a missense mutation but stays silent
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