Cloning sgRNA

I need to insert sgRNA into AIO-mCherry vector (74120-Addgene), however oligos were designed with complementary sequence for BamHI and ClaI, instead BbsI and BsaI, respectively. With this design, sgRNA are 12nt separated of scaffold RNA and I would like to know if Cas9 can induce RNP complex in order to cut target or not?

Someone can help me?

Thanks!


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Pink Werewolfover 3 years ago

Unfortunately, I do not think it would be wise to use BamH1 and Cla1 instead of the BbsI and BsaI for cloning. The reason is that the sgRNA site is built around the tracrRNA, and the sgRNA sites are designed so that your guide inserts at the correct point with the tracrRNA using Bbs1 and Bsa1. By using BamH1 and Cla1, you will introduce extra nucleotides which could affect your guide specificity. If you look at the 2016 scientific reports paper on these plasmids (senior author was Steve Jackson), they have the guide design specific in the supplementary information (table S1, https://media.nature.com/original/nature-assets/srep/2016/160415/srep24356/extref/srep24356-s1.pdf). So your forward guide should have in the 5' -> 3' direction, 'accg' then the rest of your specific guide. The reverse complementary should have, in the 5'-3' direction, 'aaac' then the rest. I would suggest using IDT for your guide oligos, they are cheap (about 5-7 canadian dollars per oligo) and the turn around time is very quick. I usually get my guides in 2 days of ordering.

One tip for the sequencing is to use the primer suggested in the paper, then look for the reverse complement sequence in your results. The primer suggested by the paper sequences the complementary strand.

I hope this helps.

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