| Summary: | Yes === Integrating a desired DNA sequence into the yeast genomes is a widely-used
genetic manipulation in the budding yeast Saccharomyces cerevisiae. The
conventional integration method is to use an integrative plasmid such as pRS
or YIplac series as the target DNA carrier. The nature of this method risks
multiple integrations of the target DNA and the potential loss of integrated DNA
during cell proliferation. In this study, we developed a novel yeast integration
strategy based on the widely used CRISPR-Cas9 system and created a set of
plasmids for this purpose. In this system, a plasmid bearing Cas9 and gRNA
expression cassettes will induce a double-strand break (DSB) inside a
biosynthesis gene such as Met15 or Lys2. Repair of the DSB will be mediated
by another plasmid bearing upstream and downstream sequences of the DSB
and an integration sequence in between. As a result of this repair the sequence
is integrated into genome by replacing the biosynthesis gene, the disruption of
which leads to a new auxotrophic genotype. The newly-generated auxotroph
can serve as a traceable marker for the integration. In this study, we
demonstrated that a DNA fragment up to 6.3 kb can be efficiently integrated
into the Met15 or Lys2 locus using this system. This novel integration strategy
can be applied to various yeasts, including natural yeast isolated from wild
environments or different yeast species such as Candida albicans. === This work was supported by the Wellcome Trust [202062 to B.H. and 207127 to A.M.] This work was also supported by a SURE studentship from the University of Sheffield [325537] awarded to P.W.D
|
|---|
