{"doi":"10.1101/2023.09.18.558350","title":"Higher-order combinatorial chromatin perturbations by engineered CRISPR-Cas12a for functional genomics","abstract":"Abstract Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting 1-3 genomic sites per cell. To develop a tool for higher-order ( > 3) combinatorial targeting of genomic sites with CRISPRi in functional genomics screens, we engineered an Acidaminococcus Cas12a variant -- referred to as mul tiplexed transcriptional interference AsCas12a (multiAsCas12a). multiAsCas12a incorporates a key mutation, R1226A, motivated by the hypothesis of nicking-induced stabilization of the ribonucleoprotein:DNA complex for improving CRISPRi activity. multiAsCas12a significantly outperforms prior state-of-the-art Cas12a variants in combinatorial CRISPRi targeting using high-order multiplexed arrays of lentivirally transduced CRISPR RNAs (crRNA), including in high-throughput pooled screens using 6-plex crRNA array libraries. Using multiAsCas12a CRISPRi, we discover new enhancer elements and dissect the combinatorial function of cis-regulatory elements. These results instantiate a group testing framework for efficiently surveying potentially numerous combinations of chromatin perturbations for biological discovery and engineering.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":393639,"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":4,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9483,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1167713,"name":"Caroline Wilson","orcid":"0000-0002-2553-4151","position":1,"is_corresponding":false},{"id":643423,"name":"Nicholas A. Sambold","orcid":null,"position":2,"is_corresponding":false},{"id":1167714,"name":"Rui Dai","orcid":"0000-0001-9905-9798","position":3,"is_corresponding":false},{"id":872268,"name":"QiLiang Chen","orcid":"0000-0002-9588-1423","position":4,"is_corresponding":false},{"id":1167715,"name":"Sara Misiukiewicz","orcid":"0000-0003-2021-1713","position":5,"is_corresponding":false},{"id":796454,"name":"Abolfazl Arab","orcid":"0000-0002-0024-2937","position":6,"is_corresponding":false},{"id":621661,"name":"Noam Teyssier","orcid":"0000-0003-4001-6296","position":7,"is_corresponding":false},{"id":872982,"name":"Thomas O’loughlin","orcid":"0000-0002-4783-2352","position":8,"is_corresponding":false},{"id":267755,"name":"Joshua C. Cofsky","orcid":"0000-0001-5403-8555","position":9,"is_corresponding":false},{"id":260600,"name":"Junwei Shi","orcid":"0000-0002-8427-6316","position":10,"is_corresponding":false},{"id":319827,"name":"Luke A. Gilbert","orcid":"0000-0001-5854-0825","position":11,"is_corresponding":false},{"id":757620,"name":"Chris C.‐S. Hsiung","orcid":"0000-0002-1366-6069","position":0,"is_corresponding":true}],"reference_count":115,"raw_metadata":null,"created_at":"2026-07-19T01:19:05.913428Z","pmid":"37781594","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":[]}