Hello,
So my query is for knocking down a gene. Will one guide serves the purpose, or does it require more than one guide RNAs in the CRISPR-Cas12a system in bacteria?
Depends on your bacteria. The majority do not have NHEJ so one should be enough. If yours does have NHEJ and you plan to knockout without recombination you might need two, however, since cpf1 cuts downstream of the PAM site, one should be theoretically work as well.
Thanks, Hanna, for responding. My bacteria has a robust NHEJ pathway. And recombination is involved, so will one crRNA is good enough, or will two be required?
My suggestion would be try it with one and if you confirm you got the KO you then you do not have to bother with two.
Ok. I will try that and see. Also, I wanted to ask if the crRNA for gene knockout will work efficiently if the guide is at the beginning of the gene (I am attaching a pdf of where the guide is highlighted in yellow). It is not in mid-part or anywhere but to the start. And what if there is only one guide RNA in the gene?
I appreciate you taking the time to write to me. It means a lot. Thank you.
Usually cut near the start gives you a better chance of a proper KO since it has a higher chance to cause an early frameshift. If you have only one guide then I would sequence multiple clones as the repair is not identical in all of them and some will be better than others. The only downside if you only have one, is that some sequences simply do not work efficiently and hence multiple guides are usually designed. As I said above the best option for you right now is to try and see.
Sure, Mam, I will try and check for multiple clones. Thank you very much for your time and suggestions.
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