Dear all,
We have recently performed CRISPR Cas9 knocking out our gene of interest in the U87 glioma cell line. We used the pX459 plasmid from Addgene and tested various gRNAs (always only one type of gRNA at a time). We selected clones based on the absence of the protein product and performed sequencing after subcloning of the PCR product in bacteria. We detected only one type of indel in all four analyzed clones (sequencing 4-5 bacterial colonies for each clone, different indels in different clones). We further directly sequenced the PCR product in 2 of the most promising clones and obtained a clear sequence showing the expected indel in the targeted region. We performed the mismatch-cleavage assay in one of these clones, which showed no cleavage, further supporting that we have the same indel in both alleles.
We are happy to have these nice homozygous knockouts, but it makes me wonder why there are not two different genetic changes in our cells? We confirmed that the cells have 2 alleles of the target gene, to my knowledge the usual type of repair for the double strand breaks should be error prone NHEJ. What is the variability of indels you obtain/would expect after CRISPR Cas9 cleavage in a cancer cell line?
Thanks for your thoughts, Petr.
The amplicon has approximately 600bp, the guide suquence is more less in the middle. Thanks for the comments! Petr
We obtained this kind of homozygous clone before, a single nucleotide deletion. After extensive characterization, we believed it is a true KO. My guess is that one allele was cleaved and repaired first, then the repaired sequence served as template for repair when the second allele was cleaved. No idea how to verify this.
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