I want to prevent expression of my gene of interest in human cell lines (eg HeLa), either by indels using a single plasmid system, or using a donor plasmid for homologous repair to insert a GFP/puro marker, such as this system http://www.origene.com/CRISPR-CAS9/
My question is, how can you interpret the (final, functional) results in CRISPR-edited cells? As an un-cloned heterogeneous population, some cells will be un-edited and many will only be edited in one allele, so in this sense, is CRISPR any better than siRNA or shRNA?
If I do single cell cloning and select for clones in which my gene is edited, even in both alleles, how can I be sure that any functional changes are specific to the CRISPR editing and not to single cell cloning itself? I have previously made single cell clones (no CRISPR, just limiting dilution of a cell line) and the resulting clones were very different in several functional assays and in levels of several surface markers. Is the only option to compare multiple edited clones to multiple negative control clones?
Thanks!
One approach is to have two donor plasmids: one with puro resistance and the other with a different such as neomycin. Then you can double select and know that all surviving cells have both alleles knocked out. Alternatively you can design A set of three PCR primer pairs. One pair will only amplify WT gene, one will have a primer for upstream of the insertion of puro cassette and the reverse primer in the cassette itself, and the third will have a forward primer in the cassette and a reverse primer downstream of insertion in your gene. If you do clinal selection you can use this to detect single allele or double allele knockouts.
Do you know the ploidy of the your target chromosome? It's likely more than two in HeLa. The answer may inform your decision, as 5 would be significantly uglier than 3 if you're attempting to hit them all separately.
If you decide to clone, I don't think there's an approach that gets around potential founder effects other than the use of multiple clones. You'll have to collect more than a single clones as part of the processes anyway, so using a few in parallel either up front for characterization or for validation of downstream assays makes sense. Especially in HeLa (because they're so variable from lab to lab and not 'well-defined' in many ways). You may also want to consider that the founder effects attributable to the CRISPR targeting sequence itself (e.g., GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases) may be present in many/all of the clones, and consider the use of a second targeting sequence (and second set of clones). Perhaps the extent to which your system of interest is already understood is relevant here - do you have strong evidence for the expected effect?
Hi, thanks for the feedback. I don't know the ploidy of my target chromosome, it is a microglial cell line that is not very commonly used. I think multiple clones (and maybe two target sequences) are the only way to go. Cheers.
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