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 converting and combining multiple loci, thus identifying compatible genotypes. Finally, we establish MERGE proficiency by engineering a fungal carotenoid biosynthesis pathway and most of the human α-proteasome core into yeast. Therefore, MERGE lays the foundation for scalable, combinatorial genome editing in yeast.
Original language | English (US) |
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Article number | 100464 |
Pages (from-to) | 100464 |
Journal | Cell Reports Methods |
Volume | 3 |
Issue number | 5 |
DOIs | |
State | Published - May 22 2023 |
Keywords
- CP: Molecular biology
- CP: Systems biology
- CRISPR-Cas9
- combinatorial genome editing
- gene drive
- humanized proteasome
- humanized yeast
- Humans
- Homologous Recombination
- Saccharomyces cerevisiae/genetics
- Gene Editing
- CRISPR-Cas Systems/genetics
- Genetic Engineering
ASJC Scopus subject areas
- Genetics
- Biochemistry, Genetics and Molecular Biology (miscellaneous)
- Biochemistry
- Radiology Nuclear Medicine and imaging
- Biotechnology
- Computer Science Applications