I'm considering designing a CRISPR guide RNA expressing plasmid with multiple sgRNAs run off one promoter. Do I need to put a direct repeat sequence in between these, RE sites, nothing, or something else entirely?
From background reading, I see others have done this, but I haven't been able to find an explanation for the plasmid set up. (ex"Multiplexed activation of endogenous genes by CRISPR-on..." Cheng 2013)
You can also just design oligos for your sgRNAs that contain a T7 site, then you can skip the whole cloning step and just amplify your sgRNAs from annealed oligos.
Hi Kira,
I have no idea what I'm doing and I'm still learning CRISPR too, but I read that you put in the direct repeat sequences http://www.genome-engineering.org/crispr/?page_id=159 But again, I just asked a question myself so don't take my word for it.
See here the answers: https://www.researchgate.net/post/What_are_some_efficient_methods_to_clone_multiple_guide_RNAs_into_a_CRISPR_Cas9_nickase_expression_vector
You can also just design oligos for your sgRNAs that contain a T7 site, then you can skip the whole cloning step and just amplify your sgRNAs from annealed oligos.
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