{"doi":"10.3390/genes13122363","title":"A Tet-Inducible CRISPR Platform for High-Fidelity Editing of Human Pluripotent Stem Cells","abstract":"Pluripotent stem cells (PSCs) offer an exciting resource for probing human biology; however, gene-editing efficiency remains relatively low in many cell types, including stem cells. Gene-editing using the CRISPR-Cas9 system offers an attractive solution that improves upon previous gene-editing approaches; however, like other technologies, off-target mutagenesis remains a concern. High-fidelity Cas9 variants greatly reduce off-target mutagenesis and offer a solution to this problem. To evaluate their utility as part of a cell-based gene-editing platform, human PSC lines were generated with a high-fidelity (HF) tetracycline-inducible engineered Streptococcus pyogenes SpCas9 (HF-iCas9) integrated into the AAVS1 safe harbor locus. By engineering cells with controllable expression of Cas9, we eliminated the need to include a large Cas9-expressing plasmid during cell transfection. Delivery of genetic cargo was further optimized by packaging DNA targeting guide RNAs (gRNAs) and donor fragments into a single plasmid backbone. The potential of homology-directed repair (HDR) based gene knock-in at the CLYBL safe harbor site and endogenous SOX2 and SIX6 genes were demonstrated. Moreover, we used non-homologous end-joining (NHEJ) for gene knockout of disease-relevant alleles. These high-fidelity CRISPR tools and the resulting HF-iCas9 cell lines will facilitate the production of cell-type reporters and mutants across different genetic backgrounds.","journal":"Genes","year":2022,"id":278381,"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":7,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9439,"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":328719,"name":"Melissa K. Jones","orcid":"0000-0002-7093-2351","position":1,"is_corresponding":false},{"id":881065,"name":"Devansh Agarwal","orcid":"0000-0001-8342-6119","position":2,"is_corresponding":false},{"id":951447,"name":"Diana V. De La Toba","orcid":null,"position":3,"is_corresponding":false},{"id":951448,"name":"Netra K. Kambli","orcid":null,"position":4,"is_corresponding":false},{"id":703878,"name":"Fei Su","orcid":"0000-0002-2891-0925","position":5,"is_corresponding":false},{"id":950913,"name":"Heather M. Martin","orcid":"0000-0002-8988-2446","position":6,"is_corresponding":false},{"id":950914,"name":"Ryan J. Anderson","orcid":"0000-0003-0311-9261","position":7,"is_corresponding":false},{"id":950915,"name":"Ryan Wong","orcid":"0000-0001-8055-406X","position":8,"is_corresponding":false},{"id":951449,"name":"Justin Seid","orcid":null,"position":9,"is_corresponding":false},{"id":951450,"name":"Saisantosh V. Attaluri","orcid":null,"position":10,"is_corresponding":false},{"id":267965,"name":"Melissa Chow","orcid":"0000-0002-6501-3991","position":11,"is_corresponding":false},{"id":335089,"name":"Karl Wahlin","orcid":"0000-0002-0494-8304","position":12,"is_corresponding":false},{"id":691514,"name":"Shawna L. Jurlina","orcid":null,"position":0,"is_corresponding":true}],"reference_count":45,"raw_metadata":null,"created_at":"2026-07-19T00:28:47.357993Z","pmid":"36553630","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":[]}