I plan to use one single plasmid to express Cas9, gRNA and donor DNA for homologous recombination in bacteria. My donor DNA is mutated to exclude the PAM, so Cas9 doesn't cleave it.
Once the homologous recombination ohs complete, I have some doubts:
1. Will my plasmid be intact?
2. Will an EXCHANGE of DNA happen? Like, my donor DNA with the desired mutation go into the chromosome and the original sequence go to my plasmid? If so, will Cas9 now cleave my plasmid?
I'll also try a 2 plasmids strategy, but so far, 2 transformations is not working fine for me.
Hi Luis, my main concerns are your donor and the homologous recombination, the main mechanism used to integrate your donor into the genome, rather than the plasmid.
1. Linear dsDNA is generally more effective as a donor than plasmid/circular DNA. However, linear dsDNA is also more prone to degradation, so ideally, you need a large amount (hundreds of ng to a couple ug) of linear donor for high efficiency. Otherwise, put the donor on a separate plasmid.
2. As far as I know, bacterial homology-directed repair is not that efficient, so for CRISPR in bacteria, it usually needs accompanying lambda red recombination system to promote homologous recombination, which sort of defeats the point of using CRISPR as the first go-to method for gene editing. The reason is in bacteria, lambda red recombineering is perfectly capable of accomplishing that without CRISPR-cas9. CRISPR might be necessary if you're constructing a large library or doing something that needs ultra-high efficiency. For my experiments that only required a couple of isolates per mutation type, lambda red recombineering worked just fine.
3. For your question, there is an exchange of material, but whether cas9 cuts your plasmid or not doesn't really significantly affect editing efficiency. Cas9 is there to do the cut and stimulate DNA repair, thus increasing recombination efficiency, so once it's done its job you don't need to keep the plasmid anymore.
You can read this paper for CRISPR in E. coli. I'd try out lambda recombineering alone first honestly.
Article Development of a fast and easy method for Escherichia coli g...
Thanks for your answers, Thu! I'm working with Leptospira, so they're really difficult to transform.
I'll try to transform my bacteria with a plasmid containing only the donor DNA, than grow the recombinant bacteria and transform again with a plasmid with Cas9 and gRNA. (I use electroporation).
Now, I'll also try the dsDNA as donor. Could I use wild type bacteria and electroporate them at the same time with my (cas9 gRNA) plasmid and dsDNA?
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