{"doi":"10.3389/fcell.2021.719705","title":"CRISPR-Cas12a System With Synergistic Phage Recombination Proteins for Multiplex Precision Editing in Human Cells","abstract":"The development of CRISPR-based gene-editing technologies has brought an unprecedented revolution in the field of genome engineering. Cas12a, a member of the Class 2 Type V CRISPR-associated endonuclease family distinct from Cas9, has been repurposed and developed into versatile gene-editing tools with distinct PAM recognition sites and multiplexed gene targeting capability. However, with current CRISPR/Cas12a technologies, it remains a challenge to perform efficient and precise genome editing of long sequences in mammalian cells. To address this limitation, we utilized phage recombination enzymes and developed an efficient CRISPR/Cas12a tool for multiplexed precision editing in mammalian cells. Through protein engineering, we were able to recruit phage recombination proteins to Cas12a to enhance its homology-directed repair efficiencies. Our phage-recombination-assisted Cas12a system achieved up to 3-fold improvements for kilobase-scale knock-ins in human cells without compromising the specificity of the enzyme. The performance of this system compares favorably against Cas9 references, the commonly used enzyme for gene-editing tasks, with improved specificity. Additionally, we demonstrated multi-target editing with similar improved activities thanks to the RNA-processing activity of the Cas12a system. This compact, multi-target editing tool has the potential to assist in understanding multi-gene interactions. In particular, it paves the way for a gene therapy method for human diseases that complements existing tools and is suitable for polygenic disorders and diseases requiring long-sequence corrections.","journal":"Frontiers in Cell and Developmental Biology","year":2022,"id":272815,"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":11,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9529,"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":471187,"name":"Qiong Xia","orcid":"0000-0002-3954-1862","position":1,"is_corresponding":false},{"id":472230,"name":"Qianhe Zhang","orcid":null,"position":2,"is_corresponding":false},{"id":854309,"name":"Yuanhao Qu","orcid":"0000-0002-7574-259X","position":3,"is_corresponding":false},{"id":939386,"name":"Stephen Su","orcid":"0000-0001-8922-5684","position":4,"is_corresponding":false},{"id":471185,"name":"Jason Cheng","orcid":"0000-0002-1301-276X","position":5,"is_corresponding":false},{"id":471186,"name":"Nicholas W. Hughes","orcid":"0000-0001-9004-6288","position":6,"is_corresponding":false},{"id":15696,"name":"Le Cong","orcid":"0000-0003-4725-8714","position":7,"is_corresponding":false},{"id":471184,"name":"Chengkun Wang","orcid":"0000-0002-2505-7295","position":0,"is_corresponding":true}],"reference_count":26,"raw_metadata":null,"created_at":"2026-07-19T00:27:47.926496Z","pmid":"35774104","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":[]}