{"doi":"10.1101/2022.09.02.506401","title":"Improved Genome Editing by an Engineered CRISPR-Cas12a","abstract":"ABSTRACT CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) ( cis -activity) and indiscriminately degrade single-stranded DNA (ssDNA) ( trans -activity). To investigate how the relative potency of cis - versus trans -DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a (LbCas12a) that selectively disrupt trans -activity. The resulting engineered mutant with the biggest differential between cis - and trans -DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2-18-fold depending on the genomic locus. Finally, we found that a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy of coupled rational engineering and directed evolution establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The availability of experimental and predicted structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome editing proteins.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2022,"id":301792,"datarank":0.16479184330021646,"base_score":1.0986122886681096,"endowment":1.0986122886681096,"self_citation_contribution":0.16479184330021646,"citation_network_contribution":0.0,"self_endowment_contribution":0.16479184330021646,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9508,"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":230881,"name":"Kai Chen","orcid":"0000-0002-8647-1182","position":1,"is_corresponding":false},{"id":259596,"name":"Honglue Shi","orcid":"0000-0003-3847-1652","position":2,"is_corresponding":false},{"id":620577,"name":"Elizabeth C. Stahl","orcid":"0000-0003-0111-6315","position":3,"is_corresponding":false},{"id":556964,"name":"Benjamin A. Adler","orcid":"0000-0002-7488-3040","position":4,"is_corresponding":false},{"id":849170,"name":"Junjie Liu","orcid":"0000-0002-8982-107X","position":5,"is_corresponding":false},{"id":849171,"name":"Kaihong Zhou","orcid":"0000-0001-9267-9206","position":6,"is_corresponding":false},{"id":850121,"name":"Jinjuan Ye","orcid":null,"position":7,"is_corresponding":false},{"id":2407,"name":"Jennifer A. Doudna","orcid":"0000-0001-9161-999X","position":8,"is_corresponding":false},{"id":302175,"name":"Enbo Ma","orcid":"0000-0002-4374-796X","position":0,"is_corresponding":true}],"reference_count":30,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T00:32:06.309890Z","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":[]}