I am amateur in yeast genomics, but recently i am trying to screen some ts mutants of yeast. Most of the literatures that i ve reviewed described methods using diploid shuffle or plasmid shuffle and both of them start at diploid. So my question is can i edit the haploid yeast (e.g. MATa or MATalpha) genome using homologous recombination? Or is there any comments on how to produce ts mutants using the haploid?
Thanks.
Hi Shen Wang,
Surely you can create mutants in haploid yeast by homologous recombination!! Several article will help you for using the appropriate protocol. Here are some links for the well known articles:
1. Deletion/N or C terminal tagging/modifications using one step PCR method:
Longtine et al. 1998
http://www.ncbi.nlm.nih.gov/pubmed/9717241
2. Site directed mutagenesis:
Storici et al. 2006
http://www.ncbi.nlm.nih.gov/pubmed/16793410
You will also get the details of the template plasmids in the papers.
All the best!
Yes! you can do it easily. For haploid cells you can start with Schizosaccharomyces pombe. Add 300-bp on flaking sequences on both side of the region you want to place. The flank should be the same where you want to place it. Once done with the sequence, go for the yeast transformation procedure described by Geitz et al. 1992. use linear fragment for the transformation. I hope you will get the desired results.
All the Best
Hi Shen Wang,
Yes, you can use homologous recombination to edit haploid yeast genome. However, researchers have recently started using the much useful CRISPR-Cas systems for genome editing. So please have a look at that too.
There is also a Temperature Sensitive Conditional Mutant Collection developed by the Boone-Andrews labs. Please take a look.
Find attached, some useful references.
Best wishes,
Raj
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