Rapid, scalable, combinatorial genome engineering by marker-less enrichment and recombination of genetically engineered loci in yeast.

ABSTRACT: A major challenge to rationally building multi-gene processes in yeast arises due to the combinatorics of combining all of the individual edits into the same strain. Here, we present a precise and multi-site genome editing approach that combines all edits without selection markers using CRISPR-Cas9. We demonstrate a highly efficient gene drive that selectively eliminates specific loci by integrating CRISPR-Cas9-mediated double-strand break (DSB) generation and homology-directed recombination with yeast sexual assortment. The method enables marker-less enrichment and recombination of genetically engineered loci (MERGE). We show that MERGE converts single heterologous loci to homozygous loci at ∼100% efficiency, independent of chromosomal location. Furthermore, MERGE is equally efficient at

SUBMITTER: Abdullah M 

PROVIDER: S-EPMC10261898 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature