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Rapid, scalable, combinatorial genome engineering by Marker-less Enrichment and Recombination of Genetically Engineered loci (MERGE)

Mudabir Abdullah, Brittany M. Greco, View ORCID ProfileJon M. Laurent, Michelle Vandeloo, View ORCID ProfileEdward M. Marcotte, View ORCID ProfileAashiq H. Kachroo
doi: https://doi.org/10.1101/2022.06.17.496490
Mudabir Abdullah
1Centre for Applied Synthetic Biology, Department of Biology, 7141 Sherbrooke St. W, Concordia University, Montreal, QC, Canada
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Brittany M. Greco
1Centre for Applied Synthetic Biology, Department of Biology, 7141 Sherbrooke St. W, Concordia University, Montreal, QC, Canada
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Jon M. Laurent
2Institute of Systems Genetics, NYU Langone Health, New York, USA
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Michelle Vandeloo
1Centre for Applied Synthetic Biology, Department of Biology, 7141 Sherbrooke St. W, Concordia University, Montreal, QC, Canada
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Edward M. Marcotte
3Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA
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Aashiq H. Kachroo
1Centre for Applied Synthetic Biology, Department of Biology, 7141 Sherbrooke St. W, Concordia University, Montreal, QC, Canada
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  • For correspondence: aashiq.kachroo@concordia.ca
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Abstract

Large-scale genome engineering in yeast is feasible primarily due to prodigious homology-directed DNA repair (HDR), a plethora of genetic tools, and simple conversion between haploid and diploid forms. However, 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 an approach for scalable, precise, multi-site genome editing that combines all edits into a single strain without the need for selection markers by using CRISPR-Cas9 and gene drives. First, we show that engineered loci become resistant to the corresponding CRISPR reagent, allowing the enrichment of distinct genotypes. Next, 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) in yeast. We show that MERGE converts single heterologous yeast loci to homozygous loci at ~100% efficiency, independent of chromosomal location. Furthermore, MERGE is equally efficient at converting and combining loci, thus identifying viable intermediate genotypes. Finally, we establish the feasibility of MERGE by engineering a fungal carotenoid biosynthesis pathway and most of the human α proteasome core into yeast. MERGE, therefore, lays the foundation for marker-less, highly efficient, and scalable combinatorial genome editing in yeast.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Figure S10

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Posted June 21, 2022.
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Rapid, scalable, combinatorial genome engineering by Marker-less Enrichment and Recombination of Genetically Engineered loci (MERGE)
Mudabir Abdullah, Brittany M. Greco, Jon M. Laurent, Michelle Vandeloo, Edward M. Marcotte, Aashiq H. Kachroo
bioRxiv 2022.06.17.496490; doi: https://doi.org/10.1101/2022.06.17.496490
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Rapid, scalable, combinatorial genome engineering by Marker-less Enrichment and Recombination of Genetically Engineered loci (MERGE)
Mudabir Abdullah, Brittany M. Greco, Jon M. Laurent, Michelle Vandeloo, Edward M. Marcotte, Aashiq H. Kachroo
bioRxiv 2022.06.17.496490; doi: https://doi.org/10.1101/2022.06.17.496490

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