EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9.

ABSTRACT: Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker-free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone-MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90-100% and 60-70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113-7D) and a diploid industrial strai

SUBMITTER: Jessop-Fabre MM 

PROVIDER: S-EPMC5094547 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature