I am trying to knock out a gene in 2 strains of S.cerevisiae, BY4743 and W303 with CRISPR/Cas9. I am using the same gRNA and donor DNA for both strains because I want to knock out the same gene the same way. There is no alteration in the sequence which should be complementary to the gRNA, I checked. It works with W303 nicely, but not at all with BY4743. What can be the reason?
(Both strains are haploid.)
Maybe it's a transformation efficiency difference. Do they both transform equally well?
No, BY has quite a bad transformation efficiency, but I still managed to transform the strain with both plasmids. (The Cas9 is on a plasmid and my donor DNA is on a plasmid too, the linear donor DNA is released from the plasmid within the cell. Sorry, I forgot to write this in the description...)
The 2 plasmids work in W303, but not in BY.
Ah, well, perhaps screening lots and lots of colonies will eventually get you want you are wanting. I'm guessing you need to detect the DNA change via molecular analysis, rather than by a morphology change. Is this the first time trying gene editing in this particular strain?
As a note, we were having a low transformation rate and a lack of DNA target change in a strain here too (S. pombe). In our case, successful gene editing should lead to salt tolerance. We were getting salt tolerant colonies but no gene changes. Turns out we had a low-level of wild yeast contamination in our lab strain. Go figure.
I had multiple colonies sequenced and the target gene is still there, intact, with no alterations, without any sign of the DSB by Cas9.
With traditional transformation, we managed to do some editing in this strain before too.
By sporulation, you can make a haploid strain from a diploid. That is how I have a haploid BY4743.
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