Our lab has been trying to perform knockouts of genes involved in the WNT pathway in osteosarcoma and Ewing sarcoma cell lines (HOS, SAOS2, U2OS, MHH, A4573). When we are able to successfully demonstrate knocked out expression of a protein through western blots, the cells seem to lose their knockout after several passages. We have tried monoclonal expansions to ensure we isolate a single population of knockout cells, and while these cells maintain fluorescence and puromycin resistance, they always lose their knockout phenotype. We spoke with some other researchers who had a similar problem with osteosarcoma and Ewing sarcoma cell lines, noticing that their CRISPR-Cas9 alterations only ever seem to be temporary. Since CRISPR-Cas9 is supposed to be permanent, why could this be happening?
Some cancers form extrachromosomal nuclear material which might include homologs of what you are trying to knock out that may recombine at a later date. Have you tried running PCR across your GOI and looking at the banding pattern?
@Robert Adolf Brinzer This is a great idea. However, we are initially seeing a complete knockout of the gene, leading me to believe that even if there is extrachromosomal DNA containing the GOI, it too would have been knocked out. Unless I suppose the extrachromosomal DNA was under a different regulation mechanism that repressed it until it became necessary from the chromosomal gene being knocked out. We've seen this phenomenon with numerous genes, though, so it seems strange that that would be the case.
Hey Laurel, it could be genetic compensation where cells upregulate related pathways or redundant genes. Ewing sarcoma cell lines can particularly be inherently unstable due to their cancerous nature. I don't know why it happens exactly, but if you need a more stable nock-out, you could try CRISPR-OFF (DOI: 10.1016/j.cell.2021.03.025). It should last forever!
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