Dear all,
I'm planning to knock-out a gene of interest by using the one-vector system lenticrisprV2 and designing one sgRNA targeting a genomic region within the second exon.
My question is: if the tool works and I will achieve the KO of the gene, can I then transiently re-express the gene for a recovery experiment by using for instance a plasmid encoding the gene cDNA? I'm afraid that the lenticrisprV2 integrated in the genome of the cells will cut also any plasmid encoding for the gene?
I hope I was clear enough, and I'm looking forward for your suggestions.
Thank you in advance.
Giuseppe
If I've understood correctly you want to revert back to wildtype to ensure that the results are you are seeing were directly a result of the Crispr induced change?
I've spoken to people about this yes this is possible again through the use of Crispr but it needs to be designed specifically to revert that SNP back to its original state.
there are few options here. you can use a homolog from another species, lets say human vs mouse and vice versa or make the cDNA rescue sequence resistant to gRNA by making few nucleotide changes in that region that won't change the AA sequence.
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