So what is the purpose of the point mutation? Are you trying to knock out the gene and replace it with a mutated allele?

Precisely Robert Adolf Brinzer. I said knockdown but I meant knockout, sorry.

I think the rationale is that your selective marker would either select for clones that have had your knockdown allele inserted or maintain the plasmids with the CRISPR so increasing numbers of cells would become knockdowns as time progresses. Still probably best to just select individual clones.

By the sound of it, it's a knockdown, not a knockout. Since the CRISPR will perform mono-allelilc knockouts on most of the cells (meaning that the other allele will be able to produce protein). Therefore, unless the genes are known to have haploinsufficiency, you will get a population of cells with a knockdown and a band will still be visible but with less intensity in the westerns.

I agree with you, to achieve a full knockout you will need to keep the cells in culture and isolate pure colonies. Then, check isolated colonies until you find one with a biallelic knockout (will be rare but most likely some will have it), and then expand that colony. I think that their suggestion makes sense because you are assessing that the overall knockdown worked before progressing to cultivate the cells and isolate a pure colony which will save you time in case it didn't work in the first place.

Robert Adolf Brinzer is right, depending on the CRISPR editing procedure the rationale would change. If they made an insertion of the selective marker in your gene of interest to knock it out then you are just selecting for colonies with the mutation. But if they transfected a CRISPR plasmid that also conferred the cells antibiotic resistance independent in the editing, then cells will become knockdowns as time progresses with appropriate selection, but this is a bit of a dirty technique since CRISPR will also have off-target effects

Regarding your point of seeing a band at the right size that has loss function, it is incredibly unlikely that you will cause knockout by indels through loss of function while maintaining the right size. You will expect complete loss of expression or a protein of different size due to truncation.

Thank you Bruno Salomone Gonzalez de Castejon

I think I understand their intentions now. However, considering the time it has been taking for some of the antibodies to arrive and the work it has been taking to standardize the blotting, I feel like I could have done the single clones by now, which I believe I will have to do regardless.

Also, as far as I know, the resistance marker was not inserted in the region of the knockout.

Finally, do you believe a western blot of the pool is still the best strategy? One of the antibodies will still take some time to arrive.

To be honest I would perform limited dilution cloning now, so yh, make single clones or at least more diluted ones!

I would have at least diluted my cells to some extent before the western

I see! I was wondering if diluted clones were a thing, I was a bit ashamed to ask.

Thank you very much!

But maybe isolate genomic DNA and try PCR with Sanger sequencing or T7EI assessments rather than western since you will have a lot of wells (aka clones) to select from. Then perform western on good candidates