So, I've used the CRISPR P 2.0 software to look up guide RNAs to use to delete a region in my gene's promoter. Past members of our lab were using one guide RNA taken straight from that website to make one primer, and another guide RNA but reverse complemented to make the other primer. However, does the orientation of the guide RNA matter ?
hello Malaika Gomes ,
There should not be any problem with choosing the strand either sense or antisense since the cut is made on both the strands (DSB) follow the link https://www.addgene.org/crispr/cut/. However, for a gene, you may consider if you want a heterozygous cut or homozygous cut . get some help from this link https://www.nature.com/articles/nature17664. all the best
Hi Malaika, depends on what are you doing. If is is just a KO, orientation probably - does not matter as long as you have equally good sgRNAs. Of cause, you should have PAM(s) for both orientations ( I am not sure I am correctly understand your Q.) But in some cases, for example using oligonucleotides for KI - it does matter what strand you are targeting. That what, my understanding, latest data shows.
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