Within my project I want to use CRSIPR Cas9 to generate stable knock-in cell lines. The goal is to knock in a gene with a point mutation which leads to a single amino acid substitution. I read a lot and found that the PAM sequence should be near the point mutation. But in case of my gene there is no PAM sequence near the point mutation. So this strategy is not working.
Does someone know if I can knock out the endogenous Protein via CRISPR Cas9 and then knock in the mutated form? Or can I exchange the exon where the point mutation is located?
Maybe someone had the same problem and has an idea how I can manage this?
Many thanks in advance
The efficiency of HDR, just like homologous recombination, is dependent on the distance between your knock-in mutation to the dsDNA breaks. The closer, the higher efficiency. If you can find a crispr site 100 bp within your intended point mutation, you should still be able to get a knock-in. In this case, you probably can not use ssOligo as a donor instead use plasmid donor with 500-1000 bp homolog arms. You may need to modify the donor dna sequence not to have the crispr site by silent mutations.
Alternatively, you can knock out your GOI and overexpress a mutant gene by pBabe retrovirus stable expression (you can screen for single clones with expression level similar to endogenous level)
My strategy "a sort of endogenous tagging in the middle"
I have recently built a knock-in cell line with the same problem you encountered.
Though I am not sure the location of your desired mutation, you still can choose the upstream of your desired mutation with Cas9 (in my case more than 150 bp) and design a HDR vector which includes the region from the upstream of the cut site all the way down to stop codon and poly A tail, including your mutation of your gene. Thus the expression of GOI is driven from the endogenous promoter but achieved from this hybrid sequence, which disrupts gene expression from the downstream of your desired mutation. Folks, does anyone have a different experience, idea.........
-Please forgive any typos. written on train with tiny phone.
Dear all,
thank you very much for your useful comments!
@ Chunxin Wang : If the sequence has to be within 100bp that´s possible. I would like to use nickase strategy. I found a PAM sequence 27bp upstream from the desired mutation and the next PAM sequence 17bp downstream. Can you give me an advise if I can use ssOligo or do I have to use a donor plasmid?
@Yves Matthess: The best would be to keep the reading frame original.
The plasmid with the gRNA contains GFP. I wanted to sort for GFP positiv single cells and after that I think I have to do DNA sequencing. Or do you have another cheaper idea?
Yes so far this is the strategy except I find a better/cheaper solution.
- Badges
- Science topic