Currently I have an experimental problem I can’t get pass. I’m supposed to knock-out a glycosyltransferase gene in a tumoral cell line using the CRISPR-Cas 9 technic. So far, I’ve tried to sort the cells of interest using a cell sorter but that was too harsh, and they died. Then, I tried to select transfected cells using the single-clone isolation protocol, but my cells simply can’t recapitulate its number from single clones and they died. Now I’m thinking in the possibility of antibiotic selection, but I understand it can only be done if you knock-in the resistance gene in the same cells. Is it possible to knock-out a gene of interest and knock-in the resistance gene at the same time? Any ideas? Of course, changing the cell line its not a possibility at the moment.
Hi there,
If the DNA fragment you propose for the repair of the gap contains a resistance cassette there should not be any problem. Then it's rather a knock-in than a knock-out.
I am guessing that by knock-out, you use the classical CRISPR/Cas9 method where you transfect the cells with Cas9 gene and also the gRNA targeting the site to create frameshift mutation. If so, during transfection, you can use vector that provide antibiotic resistance gene (puromycin etc) for selection of the cells acquiring the transfected plasmids. Basically, it is the selection of vector type (lentivirus etc) used for your transfection (refer to Addgene). But, even with antibiotic resistance, you will end up with a pool of knock-out cells, where it is hard to know if 100% of the cells are homozygously knock-out, or only a having an allele deleted, or even cells that happens to have the resistance gene but not knock-out of your target gene etc. You will still have to generate a single cell clone from the pool, but to choose the right pool, you can used any assay to check the presence of the gene (proteins or function) and choose the pool with least function of the target genes. It will imply that most of the cells are knock-out so you have higher chances of ending with a single total knock-out clone. For the dying cells, you might want to seed more cells, to allow them to sense the presence of each other while maintaining a single cell distance for isolation. This might help, https://www.researchgate.net/post/Does_anyone_have_some_experience_useful_advice_for_isolating_single_clones
Hi Andreia,
As Dominique was suggesting, the best strategy you can follow is to remove e.g. one exon from your gene of interest (GOI) and replace it with an antibiotic/fluorescent selection cassette (or alternatively, truncating one of the initial exons by the cassette insertion itself). In this case, you're generating a KO cell line via a KI strategy. Even, in case you have other selection cassettes in your cell line of interest, and you prefer to keep it as simple/clean as possible in this locus, you can always design the template DNA with the selection cassette flanked by loxP sites. Finally, as you were mentioning that your gene encodes a glycosyltransferase, I would highly recommend you to verify putative truncated isoforms with functional remaining activities to avoid undesired results after the KO generation.
Here, you'll find a very useful link from Addgene regarding those strategies:
https://blog.addgene.org/plasmids-101-knockout/knock-in-plasmids
Hope it helps!
Ruben
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