Cas9 cleaves 3 to 4 nucleotide upstream of the PAM sequence. So, it is hard to say that your construct will work and u can use the vector pspcas9 bb-2a-gfp (px458).
In theory it should work but it might be tricky getting them both to translate efficiently. If you're working in mammalian cells, the vector that Ankit suggest would work, as the the Cas9 and GFP ORFs are separated by a self-cleaving peptide region called 2A. The following publication might be helpful:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084703/
Since other researchers have made this self-cleaving Cas9-GFP you should be able to recreate it yourself pretty easily, in whatever vector you want. The sequence for pSpCas9(BB)-2A-GFP is on addgene (https://www.addgene.org/48138/) and the original publication appeared in Nature Protocols: https://www.nature.com/nprot/journal/v8/n11/full/nprot.2013.143.html
Hi Skyler,
I have designed and used gRNA construct for gene KO for mammalian cell line. If you refer literatures, they all recommend to target first few exons rather than 5'UTR or farther exons. Targeting farther exons might create transcript variants.
The scenario you mentioned above might be efficient for transcription downregulation or truncated product. Personally this rationale is not recommended.
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