Hi,
Does anyone have experience in RNA editing with CRISPR Cas9 method? Does this tool have application limited only to gene editing or I am wondering whether intergenic regions that code for small regulatory RNAs can be targeted? I feel if employed in the latter case, this technique can result still in production of sRNA with a single nucleotide indel. Kindly advise.
CRISPR/Cas can be used to target any place in the genome where you have a PAM sequence. Indel size vary so if a single nucleotide deletion is not sufficient for your needs than you can screen for clones with larger deletions. in cases of micro RNA's a single nucleotide deletion in the seed sequence may be enough. Alternatively you can use 2 sgRNAs and create a larger deletion that will remove your entire small RNA region.
Hej Siva,
in theory, you could target any portion of DNA you like. However, limitations to the available CRISPR systems (like specific PAM sequences in the vicinity of your target, production of rather large Indels depending on the system...) and accessibility of your region for a protein like Cas9 (160 kDa) may render your target region difficult to manipulate - at least if you want to do it yourself. Also (depending on the cells your are using), I would not underestimate the issue of efficiently delivering pDNA or ribonucleoprotein into cells - I've been trying to stuff two plasmids into hard-to-transfect-cells for a year now and it's just not happening.
Maybe there is a way to employ RNAi for your purposes?
Cheers!
It is possible, but you will have a lot of off-targets, as the microRNAs are very short sequence. You may try the Nick system using two separate sgRNA. It will help you to avoid the off-targets and unwanted clones.
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