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

Mudabir Abdullah, Brittany M. Greco, Jon M. Laurent, Riddhiman K. Garge, Daniel R. Boutz, Michelle Vandeloo, Edward M. Marcotte, Aashiq H. Kachroo

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

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 languageEnglish (US)
Article number100464
Pages (from-to)100464
JournalCell Reports Methods
Volume3
Issue number5
DOIs
StatePublished - 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

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