We are genome editing Clotridioides difficile via CRISPR system. However, we met some difficulties generating a clean knock out. The colonies after editing showed both WT and KO PCR bands as the attached gel shows. We tried to re-streak and isolate single colonies multiple times, but we always got WT colonies instead (couldn't isolate KO colonies). Would you please give us some suggestions about this issue? Thanks a lot!
Using CRISPR genome editing to generate mixed colonies of Clostridioides difficile (formerly known as Clostridium difficile) could be a challenging task due to the complexity of the organism and the intricacies of CRISPR technology. However, it's theoretically possible with careful design and execution.
Here's a general outline of how you might attempt this:
It's important to note that generating mixed colonies of C. difficile using CRISPR genome editing would require careful optimization of experimental conditions and rigorous validation of the resulting colonies.
Ji Zeng First of all, I would like to know whether the lane result (showing two PCR bands (WT and KO bands) at the same time) shown in your attached gel can be repeated? That is to say, from the original clone colony, pick out to do the colony PCR, can you repeat the results of your attached gel? If not, your PCR operation is wrong, resulting in a false positive; If you can repeat it, well, keep that single colony, and cut down the KO band, and sequence it to determine whether your so-called KO band is really a KO band. In addition, I suggest you can redesign the identification primer to verify whether it can produce the same results.
In addition, it gives me the feeling that, in general, when PCR identification is done in a clone, it is rarely said that there is both WT and KO bands. I feel that this lane result is a false positive, or exist mistakes in your PCR operation, resulting in there is the KO band we want, but actually it is not. In addition, I suggest adding more accurate DNA markers (that is, choosing markers that are more consistent with your ideal KO band size, so as to facilitate the identification of whether the size is correct). In addition, I also suggest that you redesign the identification primers for verification.
There's another possibility, from your description, that it feels like it's possible that the bacterial genome contains multiple target genes that haven't been knocked out completely? If so, there are multiple target genes in the bacterium, it is recommended that the experimenter take a detailed look at the C. difficile genome to make sure that there is only one target gene in the whole genome. If there's only one target gene in the genome, basically, it won't happen that both WT and KO bands exist simultaneously when you do PCR validation. If there are multiple target genes in the genome, the design of sgRNA should be more rigorous. For details, refer to the principles of sgRNA design.
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