{"doi":"10.1101/2020.07.01.183194","title":"Catalytically Enhanced Cas9 through Directed Protein Evolution","abstract":"Abstract The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 system has found widespread applications in genome manipulations due to its simplicity and effectiveness. Significant efforts in enzyme engineering have been made to improve the CRISPR-Cas9 systems beyond their natural power with additional functionalities such as DNA modification, transcriptional regulation, and high target selectivity 1–10 . Relatively less attention, however, has been paid to improving the catalytic efficiency of CRISPR-Cas9. Increased catalytic efficiency may be desired in applications where the currently available CRISPR-Cas9 tools are either ineffective 4, 11–14 or of low efficiency such as with type II-C Cas9 15–18 or in non-mammals 19, 20 . We describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9). We demonstrate the effectiveness of this method with a previously characterized Type IIC Cas9 from Acidothermus cellulolyticus (AceCas9) with up to 4-fold improvement of in vitro catalytic efficiency, as well as the widely used Streptococcus pyogenes Cas9 (SpyCas9), which showed a 2-fold increase in homology directed repair (HDR)-based gene insertion in human colon cancer cells.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2020,"id":123845,"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":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9482,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":567368,"name":"Mitchell O. Roth","orcid":"0009-0002-4948-6032","position":1,"is_corresponding":false},{"id":483687,"name":"Chardasia L. Smith","orcid":null,"position":2,"is_corresponding":false},{"id":567369,"name":"Emily Shiel","orcid":"0009-0009-6491-4331","position":3,"is_corresponding":false},{"id":319636,"name":"Kyle N. Klein","orcid":"0000-0002-7713-2787","position":4,"is_corresponding":false},{"id":42992,"name":"David M. Gilbert","orcid":"0000-0001-8087-9737","position":5,"is_corresponding":false},{"id":482420,"name":"Hong Li","orcid":"0000-0003-2046-9861","position":6,"is_corresponding":false},{"id":482418,"name":"Travis H. Hand","orcid":"0000-0002-4923-9165","position":0,"is_corresponding":true}],"reference_count":37,"raw_metadata":null,"created_at":"2026-07-18T23:15:03.403566Z","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":[]}