I'm using the CRISPR/Cas9 gene editing method to target a gene in human iPSCs for mutation correction via the homology directed repair pathway. I want to compare the efficiency of different gRNAs to determine which gRNA will be the best to use for this application. Normally, this can easily be done using a genomic cleavage detection kit or a mismatch detecting endonuclease, followed by visualization of the resulting fragments on a gel; however, the mutation we are trying to correct is heterozygous in this particular patient. Since mismatch endonucleases, such as the surveyor nuclease, are sensitive enough to detect a single bp change, would the heterozygous nature of this SNP lead to a overestimate of the level of cleavage occurring?
Has anyone else come across this issue? If so, does anyone have suggestions for other methods to detect cleavage events?
Your choice of guide RNA is going to be limited since you need to be close to the point mutation so that the DSB is very close to the site you wish to correct. Any guide RNA will likely create a DSB on both alleles. In any case it seems simple enough to transfect your guide RNA together with the repair template and clone out the cells using a selectable marker within the cas9/gRNA plasmid. Then do PCR across the region and sequence the results. You will have either a perfect sequence of the correct type or you will get double bands once you hit the deletion and/or point mutations.
Hi Gregory,
Thank you for your answer. We have already designed several gRNAs and we want to determine which one will give us the best targeting and cleavage efficiency prior to introducing the correction template for HDR. We are using an ssODN correction template instead of a CRISPR plasmid, so selection using a selection marker will not be possible. We only want to ensure that this heterozygosity is not going to affect the surveyor nuclease assay prior to the application of our ssODN correction template.
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