{"doi":"10.1016/j.jbc.2021.100990","title":"A Cas12a-based CRISPR interference system for multigene regulation in mycobacteria","abstract":"Mycobacteria are responsible for a heavy global disease burden, but their relative genetic intractability has long frustrated research efforts. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) has made gene repression in mycobacteria much more efficient, but limitations of the prototypical Cas9-based platform, for example, in multigene regulation, remain. Here, we introduce an alternative CRISPRi platform for mycobacteria that is based on the minimal type V Cas12a enzyme in combination with synthetic CRISPR arrays. This system is simple, tunable, reversible, can efficiently regulate essential genes and multiple genes simultaneously, and works as efficiently in infected macrophages as it does in vitro. Together, Cas12a-based CRISPRi provides a facile tool to probe higher-order genetic interactions in mycobacteria including Mycobacterium tuberculosis (Mtb), which will enable the development of synthetically lethal drug targets and the study of genes conditionally essential during infection. Mycobacteria are responsible for a heavy global disease burden, but their relative genetic intractability has long frustrated research efforts. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) has made gene repression in mycobacteria much more efficient, but limitations of the prototypical Cas9-based platform, for example, in multigene regulation, remain. Here, we introduce an alternative CRISPRi platform for mycobacteria that is based on the minimal type V Cas12a enzyme in combination with synthetic CRISPR arrays. This system is simple, tunable, reversible, can efficiently regulate essential genes and multiple genes simultaneously, and works as efficiently in infected macrophages as it does in vitro. Together, Cas12a-based CRISPRi provides a facile tool to probe higher-order genetic interactions in mycobacteria including Mycobacterium tuberculosis (Mtb), which will enable the development of synthetically lethal drug targets and the study of genes conditionally essential during infection. The adaptive bacterial immune systems based on clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have transformed genetic manipulation, and the ease with which they can be programmed has led to their wide use in gene editing in eukaryotes and prokaryotes (1Knott G.J. Doudna J.A. CRISPR-Cas guides the future of genetic engineering.Science. 2018; 361: 866-869Crossref PubMed Scopus (500) Google Scholar). One application of the CRISPR/Cas system, CRISPR interference (CRISPRi), introduced a new way of gene regulation by coexpressing an inactive Cas9 nuclease with an engineered single guide RNA (sgRNA) that directs the inactive nuclease to a target gene where it blocks transcription rather than cleaves the DNA (2Qi L.S. Larson M.H. Gilbert L.A. Doudna J.A. Weissman J.S. Arkin A.P. Lim W.A. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.Cell. 2013; 152: 1173-1183Abstract Full Text Full Text PDF PubMed Scopus (2538) Google Scholar). The prototypical CRISPRi system is based on an inactive type 2-II Cas9 nuclease (dCas9) and has recently also been adapted for use in mycobacteria including Mtb (3Choudhary E. Thakur P. Pareek M. Agarwal N. Gene silencing by CRISPR interference in mycobacteria.Nat. Commun. 2015; 6: 6267Crossref PubMed Scopus (132) Google Scholar, 4Rock J.M. Hopkins F.F. Chavez A. Diallo M. Chase M.R. Gerrick E.R. Pritchard J.R. Church G.M. Rubin E.J. Sassetti C.M. Schnappinger D. Fortune S.M. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform.Nat. Microbiol. 2017; 2: 16274Crossref PubMed Scopus (163) Google Scholar, 5Singh A.K. Carette X. Potluri L.P. Sharp J.D. Xu R. Prisic S. Husson R.N. Investigating essential gene function in Mycobacterium tuberculosis using an efficient CRISPR ","journal":"Journal of Biological Chemistry","year":2021,"id":178585,"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":19,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9516,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":724441,"name":"Christoph Grundner","orcid":"0000-0003-0596-6798","position":1,"is_corresponding":false},{"id":724440,"name":"Neil Fleck","orcid":"0000-0001-8327-4688","position":0,"is_corresponding":true}],"reference_count":29,"raw_metadata":null,"created_at":"2026-07-18T23:47:40.592580Z","pmid":"34298016","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":[]}