Hi Everyone. I want to use CRISPR-Cas9 system to knock in two mutations of one gene in cells, but I would like these two mutations occur on different alleles, i.e. one allele carries one mutation and the other allele carries the second mutation but on the same gene. I can image it's not difficult to make it in mouse model, but how can I detect this compound heterozygous in cell line? For example, how can I distinguish from the two mutations on the same allele and the other allele is intact? Thank you!
I guess, it is two mutations in the same locus but at different spots which are too far apart to be on the same sequencing template? The easiest way to generate these, is to do dilution cloning on double mutant pool and then screen for clones with presence of both mutations. Than you characterize each clone for heteroallelic mutations. If the distance less than 10kb you could do a long PCR (out-out PCR), clone them and sequence region of interest to fish out clones with mutations on different alleles. If the distance more than that, than you will likely have some SNPs which you could use to distinguish between alleles and as such, again, after cloning, - figure out which allele has a mutation or both mutations etc. If you are lucky, and your mutations are close enough for long (short) PCR - than, one can try to design double-mutant specific primers (where your primers has 3' ends with your mutant bases) and filter out the same allele doubles easily. In any case presence of mutations on both alleles in both spots could be a frequent complication, So it might be quicker to get a one mutated allele clone first and than introduce second mutation and screen for separate allele double mutant as above. It might be safer to go this way, cause cytotoxicity from 2 CASPR/Cas9 off-targets might be a problem. Hope it helps.
thanks, Dmitry, very good suggestions. we will try to make one mutation first and then the other one. unfortunately the two spots are far apart to each other, as you suggested, we will try to find the nearest SNPs and do PCR.
I would design the knockin to specifically introduce or remove a (rare?) restriction site. This would allow for you to map the mutations to either same or different alleles by Southern blot analysis.
Best of luck
Great Idea, Kristian! Haven't occur to me ;). I even think that simple PCR and the 50% restriction presence will work, to avoid going all the way to Southern. That is of cause if introduction of "extra" mutation is possible, which is unlikely, judging by careful experimental design. :)
Yuming, if introduction of silent mutations somewhere around one or both mutated sites with restriction sites knockin is a possibility, then Kristian's scheem is a great option. Simply doing two PCR and observing 50% restriction on each site is an easy way to screen for single knockin at each site and than doing SNP discrimination or Southern if distance is less than say 20 kb is a good trick. Kristian correct me if I am not thinking strait.
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