{"doi":"10.1101/2022.06.17.496490","title":"Rapid, scalable, combinatorial genome engineering by Marker-less Enrichment and Recombination of Genetically Engineered loci (MERGE)","abstract":"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 M arker-less E nrichment and R ecombination of G enetically E ngineered 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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2022,"id":303634,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9456,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":996081,"name":"Brittany M. Greco","orcid":null,"position":1,"is_corresponding":false},{"id":343914,"name":"Jon M. Laurent","orcid":"0000-0001-6583-4741","position":2,"is_corresponding":false},{"id":996082,"name":"Michelle Vandeloo","orcid":null,"position":3,"is_corresponding":false},{"id":240136,"name":"Edward M. Marcotte","orcid":"0000-0001-8808-180X","position":4,"is_corresponding":false},{"id":343918,"name":"Aashiq H. Kachroo","orcid":"0000-0001-9770-778X","position":5,"is_corresponding":false},{"id":996080,"name":"Mudabir Abdullah","orcid":null,"position":0,"is_corresponding":true}],"reference_count":43,"raw_metadata":null,"created_at":"2026-07-19T00:32:24.307938Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}