I am currently using the CRISPR base editing system in bacteria. Unfortunately, I'm not getting any clones with edited bases (not even off-target) but i'm not sure why. I'm quite sure the plasmid has been successfully transformed into the bacterial cell but I keep getting negatives. I've even checked my design and and my experimental procedures but I'm not sure what's wrong. Does anyone have any suggestions or is there anyone I can consult regarding this matter?
are you getting A lot of clones that aren't correct or simply an empty plate? If you are getting clones that aren't correct then I would suggest to design a new guide usually up to 3 guides are designed (using online gRNA software) since somtimes guides simply fail to cut and the reason for that is not fully known. If you are not getting anything at all I suggest you check your repair template as presence of mutations in there can reduce the rate of recombination.
Hanna Alalam Hi, Hanna! Thanks for your response. I'm actually getting a lot of clones that "aren't correct". Basically, all my clones are negatives. I'm actually getting my plasmids into Streptomyces strains through conjugation. My plates have exconjugants but none of them contain any bases edited (i.e. they're all wild type sequences when i sent them for sequencing). Also, there is no repair template used here because I'm using a base editor system that directly modifies one single nucleotide without forming DSBs.
Hanna Alalam yeah maybe i'll see if i can find more suitable guides. Thank you!
Hi Reachel.
Could you figure out what happen? I just have the same problem in pseudomonas aeruginosa.
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