{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T10:07:42Z","timestamp":1791972462087,"version":"4.3.4"},"reference-count":130,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2024,2,20]],"date-time":"2024-02-20T00:00:00Z","timestamp":1708387200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"A Cure for Robert, Inc.","award":["1R01HD102545-01A1"],"award-info":[{"award-number":["1R01HD102545-01A1"]}]},{"name":"Austrian MPS society","award":["1R01HD102545-01A1"],"award-info":[{"award-number":["1R01HD102545-01A1"]}]},{"name":"The Carol Ann Foundation","award":["1R01HD102545-01A1"],"award-info":[{"award-number":["1R01HD102545-01A1"]}]},{"name":"Angelo R. Cali and Mary V. Cali Family Foundation, Inc.","award":["1R01HD102545-01A1"],"award-info":[{"award-number":["1R01HD102545-01A1"]}]},{"name":"The Vain and Harry Fish Foundation, Inc.","award":["1R01HD102545-01A1"],"award-info":[{"award-number":["1R01HD102545-01A1"]}]},{"name":"The Bennett Foundation","award":["1R01HD102545-01A1"],"award-info":[{"award-number":["1R01HD102545-01A1"]}]},{"name":"Jacob Randall Foundation","award":["1R01HD102545-01A1"],"award-info":[{"award-number":["1R01HD102545-01A1"]}]},{"name":"Nemours Funds","award":["1R01HD102545-01A1"],"award-info":[{"award-number":["1R01HD102545-01A1"]}]},{"name":"Eunice Kennedy Shriver","award":["1R01HD102545-01A1"],"award-info":[{"award-number":["1R01HD102545-01A1"]}]},{"name":"National Institute of Child Health and Human Development of the National Institutes of Health (NICHD)","award":["1R01HD102545-01A1"],"award-info":[{"award-number":["1R01HD102545-01A1"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Since its discovery in 2012, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system has supposed a promising panorama for developing novel and highly precise genome editing-based gene therapy (GT) alternatives, leading to overcoming the challenges associated with classical GT. Classical GT aims to deliver transgenes to the cells via their random integration in the genome or episomal persistence into the nucleus through lentivirus (LV) or adeno-associated virus (AAV), respectively. Although high transgene expression efficiency is achieved by using either LV or AAV, their nature can result in severe side effects in humans. For instance, an LV (NCT03852498)- and AAV9 (NCT05514249)-based GT clinical trials for treating X-linked adrenoleukodystrophy and Duchenne Muscular Dystrophy showed the development of myelodysplastic syndrome and patient\u2019s death, respectively. In contrast with classical GT, the CRISPR\/Cas9-based genome editing requires the homologous direct repair (HDR) machinery of the cells for inserting the transgene in specific regions of the genome. This sophisticated and well-regulated process is limited in the cell cycle of mammalian cells, and in turn, the nonhomologous end-joining (NHEJ) predominates. Consequently, seeking approaches to increase HDR efficiency over NHEJ is crucial. This manuscript comprehensively reviews the current alternatives for improving the HDR for CRISPR\/Cas9-based GTs.<\/jats:p>","DOI":"10.3390\/ijms25052456","type":"journal-article","created":{"date-parts":[[2024,2,20]],"date-time":"2024-02-20T04:04:14Z","timestamp":1708401854000},"page":"2456","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":62,"title":["Current Strategies for Increasing Knock-In Efficiency in CRISPR\/Cas9-Based Approaches"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5956-1986","authenticated-orcid":false,"given":"Andr\u00e9s","family":"Leal","sequence":"first","affiliation":[{"name":"Nemours Children\u2019s Health, Wilmington, DE 19803, USA"},{"name":"Institute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogot\u00e1 110231, Colombia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Angelica","family":"Herreno-Pach\u00f3n","sequence":"additional","affiliation":[{"name":"Nemours Children\u2019s Health, Wilmington, DE 19803, USA"},{"name":"Faculty of Arts and Sciences, University of Delaware, Newark, DE 19716, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9791-2716","authenticated-orcid":false,"given":"Eliana","family":"Benincore-Fl\u00f3rez","sequence":"additional","affiliation":[{"name":"Nemours Children\u2019s Health, Wilmington, DE 19803, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amali","family":"Karunathilaka","sequence":"additional","affiliation":[{"name":"Nemours Children\u2019s Health, Wilmington, DE 19803, USA"},{"name":"Faculty of Arts and Sciences, University of Delaware, Newark, DE 19716, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0673-2160","authenticated-orcid":false,"given":"Shunji","family":"Tomatsu","sequence":"additional","affiliation":[{"name":"Nemours Children\u2019s Health, Wilmington, DE 19803, USA"},{"name":"Faculty of Arts and Sciences, University of Delaware, Newark, DE 19716, USA"},{"name":"Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu 501-1194, Japan"},{"name":"Department of Pediatrics, Thomas Jefferson University, Philadelphia, PA 19144, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,2,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"eadd8643","DOI":"10.1126\/science.add8643","article-title":"CRISPR technology: A decade of genome editing is only the beginning","volume":"379","author":"Wang","year":"2023","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"106968","DOI":"10.1016\/j.ymgme.2022.106968","article-title":"The landscape of CRISPR\/Cas9 for inborn errors of metabolism","volume":"138","author":"Leal","year":"2022","journal-title":"Mol. Genet. Metab."},{"key":"ref_3","first-page":"353","article-title":"Mechanism and Applications of CRISPR\/Cas-9-Mediated Genome Editing","volume":"15","author":"Asmamaw","year":"2021","journal-title":"Biologics"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1007\/s40291-020-00500-8","article-title":"Expansion of the CRISPR\/Cas Genome-Sculpting Toolbox: Innovations, Applications and Challenges","volume":"25","author":"Batool","year":"2021","journal-title":"Mol. Diagn. Ther."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2150","DOI":"10.1016\/j.apsb.2021.05.020","article-title":"In vivo delivery of CRISPR-Cas9 therapeutics: Progress and challenges","volume":"11","author":"Behr","year":"2021","journal-title":"Acta Pharm. Sin. B"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Kantor, A., McClements, M.E., and MacLaren, R.E. (2020). CRISPR-Cas9 DNA Base-Editing and Prime-Editing. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21176240"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1146\/annurev-biophys-062215-010822","article-title":"CRISPR-Cas9 Structures and Mechanisms","volume":"46","author":"Jiang","year":"2017","journal-title":"Annu. Rev. Biophys."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1089\/crispr.2021.0014","article-title":"Increasing Gene Editing Efficiency for CRISPR-Cas9 by Small RNAs in Pluripotent Stem Cells","volume":"4","author":"Shahryari","year":"2021","journal-title":"CRISPR J."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1007\/s00018-019-03365-1","article-title":"Structural basis of homologous recombination","volume":"77","author":"Sun","year":"2020","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3400","DOI":"10.1016\/j.molcel.2021.07.005","article-title":"Cryo-EM of NHEJ supercomplexes provides insights into DNA repair","volume":"81","author":"Chaplin","year":"2021","journal-title":"Mol. Cell"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"691","DOI":"10.3389\/fgene.2018.00691","article-title":"Methodologies for Improving HDR Efficiency","volume":"9","author":"Liu","year":"2018","journal-title":"Front. Genet."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1172\/JCI158287","article-title":"CRISPR\/Cas therapeutic strategies for autosomal dominant disorders","volume":"132","author":"Caruso","year":"2022","journal-title":"J. Clin. Investig."},{"key":"ref_13","unstructured":"FDA (2023, December 26). FDA Approves First Gene Therapies to Treat Patients with Sickle Cell Disease, Available online: https:\/\/www.fda.gov\/news-events\/press-announcements\/fda-approves-first-gene-therapies-treat-patients-sickle-cell-disease."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1093\/bfgp\/elw025","article-title":"CRISPR-Cas9 technology: Applications and human disease modelling","volume":"16","year":"2017","journal-title":"Brief. Funct. Genom."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/j.tips.2021.10.012","article-title":"CRISPR-based therapeutics: Current challenges and future applications","volume":"43","author":"Modell","year":"2022","journal-title":"Trends Pharmacol. Sci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"7729","DOI":"10.1007\/s11033-023-08659-z","article-title":"CRISPR\/Cas-mediated genome editing in mice for the development of drug delivery mechanism","volume":"50","author":"Sowbhagya","year":"2023","journal-title":"Mol. Biol. Rep."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1719","DOI":"10.1038\/s41598-023-28732-8","article-title":"Rapid clonal identification of biallelic CRISPR\/Cas9 knock-ins using SNEAK PEEC","volume":"13","author":"Singh","year":"2023","journal-title":"Sci. Rep."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"821","DOI":"10.1016\/j.stem.2019.04.001","article-title":"Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination","volume":"24","author":"Martin","year":"2019","journal-title":"Cell Stem Cell"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1709","DOI":"10.1126\/science.1138140","article-title":"CRISPR provides acquired resistance against viruses in prokaryotes","volume":"315","author":"Barrangou","year":"2007","journal-title":"Science"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1038\/s41579-019-0299-x","article-title":"Evolutionary classification of CRISPR-Cas systems: A burst of class 2 and derived variants","volume":"18","author":"Makarova","year":"2020","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1164461","DOI":"10.3389\/fpls.2023.1164461","article-title":"A comprehensive appraisal of mechanism of anti-CRISPR proteins: An advanced genome editor to amend the CRISPR gene editing","volume":"14","author":"Choudhary","year":"2023","journal-title":"Front. Plant Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1007\/s12033-021-00422-8","article-title":"CRISPR\/Cas-Based Modifications for Therapeutic Applications: A Review","volume":"64","author":"Bharathkumar","year":"2022","journal-title":"Mol. Biotechnol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1146\/annurev-genet-071719-022559","article-title":"The tracrRNA in CRISPR Biology and Technologies","volume":"55","author":"Liao","year":"2021","journal-title":"Annu. Rev. Genet."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4154","DOI":"10.1172\/JCI72992","article-title":"Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9","volume":"124","author":"Gupta","year":"2014","journal-title":"J. Clin. Investig."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"24356","DOI":"10.1038\/srep24356","article-title":"CRISPR-Cas9(D10A) nickase-based genotypic and phenotypic screening to enhance genome editing","volume":"6","author":"Chiang","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1216","DOI":"10.1038\/s41591-018-0137-0","article-title":"A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells","volume":"24","author":"Vakulskas","year":"2018","journal-title":"Nat. Med."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"6082","DOI":"10.1038\/s41467-020-19842-2","article-title":"MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits","volume":"11","author":"Ma","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"490","DOI":"10.1038\/nature16526","article-title":"High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects","volume":"529","author":"Kleinstiver","year":"2016","journal-title":"Nature"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1555","DOI":"10.1016\/j.celrep.2016.01.019","article-title":"Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair","volume":"14","author":"Gutschner","year":"2016","journal-title":"Cell Rep."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1133","DOI":"10.1038\/s41467-018-03475-7","article-title":"CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair","volume":"9","author":"Charpentier","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2866","DOI":"10.1038\/s41467-019-10735-7","article-title":"CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites","volume":"10","author":"Jayavaradhan","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1038\/s41434-019-0119-y","article-title":"Integrating gene delivery and gene-editing technologies by adenoviral vector transfer of optimized CRISPR-Cas9 components","volume":"27","author":"Maggio","year":"2020","journal-title":"Gene Ther."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1126\/science.aad5227","article-title":"Rationally engineered Cas9 nucleases with improved specificity","volume":"351","author":"Slaymaker","year":"2016","journal-title":"Science"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1038\/nature26155","article-title":"Evolved Cas9 variants with broad PAM compatibility and high DNA specificity","volume":"556","author":"Hu","year":"2018","journal-title":"Nature"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1038\/s41551-019-0505-1","article-title":"High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells","volume":"4","author":"Kim","year":"2020","journal-title":"Nat. Biomed. Eng."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1038\/s41422-018-0131-6","article-title":"Structural insights into a high fidelity variant of SpCas9","volume":"29","author":"Guo","year":"2019","journal-title":"Cell Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1038\/nature24268","article-title":"Enhanced proofreading governs CRISPR-Cas9 targeting accuracy","volume":"550","author":"Chen","year":"2017","journal-title":"Nature"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Ikeda, A., Fujii, W., Sugiura, K., and Naito, K. (2019). High-fidelity endonuclease variant HypaCas9 facilitates accurate allele-specific gene modification in mouse zygotes. Commun. Biol., 2.","DOI":"10.1038\/s42003-019-0627-8"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1380","DOI":"10.1016\/j.cell.2013.08.021","article-title":"Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity","volume":"154","author":"Ran","year":"2013","journal-title":"Cell"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"13292","DOI":"10.1038\/s41598-019-49837-z","article-title":"Nuclease dead Cas9 is a programmable roadblock for DNA replication","volume":"9","author":"Whinn","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"146518","DOI":"10.1016\/j.gene.2022.146518","article-title":"Overview of advances in CRISPR\/deadCas9 technology and its applications in human diseases","volume":"830","author":"Kanafi","year":"2022","journal-title":"Gene"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"14370","DOI":"10.1038\/ncomms14370","article-title":"Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization","volume":"8","author":"Senturk","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"656","DOI":"10.1016\/j.omtn.2022.04.013","article-title":"A dual conditional CRISPR-Cas9 system to activate gene editing and reduce off-target effects in human stem cells","volume":"28","author":"Park","year":"2022","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"542","DOI":"10.1182\/hematology.2023000487","article-title":"Gene therapy for sickle cell disease","volume":"2023","author":"Leonard","year":"2023","journal-title":"Hematol. Am. Soc. Hematol. Educ. Program."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1038\/d41587-023-00016-6","article-title":"The world\u2019s first CRISPR therapy is approved: Who will receive it?","volume":"42","author":"Sheridan","year":"2023","journal-title":"Nat. Biotechnol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"252","DOI":"10.1056\/NEJMoa2031054","article-title":"CRISPR-Cas9 Gene Editing for Sickle Cell Disease and \u03b2-Thalassemia","volume":"384","author":"Frangoul","year":"2021","journal-title":"N. Engl. J. Med."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"859","DOI":"10.1091\/mbc.E18-10-0668","article-title":"Preserving genome integrity in human cells via DNA double-strand break repair","volume":"31","author":"Jensen","year":"2020","journal-title":"Mol. Biol. Cell"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/ncb3452","article-title":"The control of DNA repair by the cell cycle","volume":"19","author":"Hustedt","year":"2016","journal-title":"Nat. Cell Biol."},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Mar\u00e9chal, A., and Zou, L. (2013). DNA damage sensing by the ATM and ATR kinases. Cold Spring Harb. Perspect. Biol., 5.","DOI":"10.1101\/cshperspect.a012716"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"389","DOI":"10.4161\/cc.9.2.10475","article-title":"The complexity of phosphorylated H2AX foci formation and DNA repair assembly at DNA double-strand breaks","volume":"9","author":"Nakamura","year":"2010","journal-title":"Cell Cycle"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"1380","DOI":"10.1128\/MCB.01639-13","article-title":"53BP1, BRCA1, and the choice between recombination and end joining at DNA double-strand breaks","volume":"34","author":"Daley","year":"2014","journal-title":"Mol. Cell. Biol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1068637","DOI":"10.3389\/fgeed.2023.1068637","article-title":"In search of an ideal template for therapeutic genome editing: A review of current developments for structure optimization","volume":"5","author":"Shakirova","year":"2023","journal-title":"Front. Genome Ed."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"582","DOI":"10.1016\/j.tig.2021.03.001","article-title":"Nonhomologous end joining: New accessory factors fine tune the machinery","volume":"37","author":"Ghosh","year":"2021","journal-title":"Trends Genet."},{"key":"ref_54","first-page":"130","article-title":"DNA double strand break repair via non-homologous end-joining","volume":"2","author":"Davis","year":"2013","journal-title":"Transl. Cancer Res."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1016\/j.dnarep.2014.02.020","article-title":"DNA-PK: A dynamic enzyme in a versatile DSB repair pathway","volume":"17","author":"Davis","year":"2014","journal-title":"DNA Repair"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"765","DOI":"10.1038\/s41580-020-00297-8","article-title":"The molecular basis and disease relevance of non-homologous DNA end joining","volume":"21","author":"Zhao","year":"2020","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"24377","DOI":"10.1074\/jbc.M116.752329","article-title":"Different DNA End Configurations Dictate Which NHEJ Components Are Most Important for Joining Efficiency","volume":"291","author":"Chang","year":"2016","journal-title":"J. Biol. Chem."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"2561","DOI":"10.1093\/nar\/gkn094","article-title":"Distinct roles of XRCC4 and Ku80 in non-homologous end-joining of endonuclease- and ionizing radiation-induced DNA double-strand breaks","volume":"36","author":"Schliecker","year":"2008","journal-title":"Nucleic Acids Res."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"971","DOI":"10.1038\/s41594-018-0133-6","article-title":"XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining","volume":"25","author":"Nemoz","year":"2018","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"24036","DOI":"10.1074\/jbc.M115.680900","article-title":"Unifying the DNA end-processing roles of the artemis nuclease: Ku-dependent artemis resection at blunt DNA ends","volume":"290","author":"Chang","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"541","DOI":"10.1016\/j.celrep.2016.09.026","article-title":"PAXX Is an Accessory c-NHEJ Factor that Associates with Ku70 and Has Overlapping Functions with XLF","volume":"17","author":"Tadi","year":"2016","journal-title":"Cell Rep."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1038\/nrm.2017.48","article-title":"Non-homologous DNA end joining and alternative pathways to double-strand break repair","volume":"18","author":"Chang","year":"2017","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"E4537","DOI":"10.1073\/pnas.1505805112","article-title":"Essential role for polymerase specialization in cellular nonhomologous end joining","volume":"112","author":"Pryor","year":"2015","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"2810","DOI":"10.1016\/j.celrep.2017.08.091","article-title":"DNA Ligase IV Guides End-Processing Choice during Nonhomologous End Joining","volume":"20","author":"Conlin","year":"2017","journal-title":"Cell Rep."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"27314","DOI":"10.1074\/jbc.M114.578823","article-title":"DNA2 cooperates with the WRN and BLM RecQ helicases to mediate long-range DNA end resection in human cells","volume":"289","author":"Sturzenegger","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"350","DOI":"10.1101\/gad.2003811","article-title":"BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair","volume":"25","author":"Nimonkar","year":"2011","journal-title":"Genes Dev."},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1016\/j.molcel.2013.04.032","article-title":"RPA coordinates DNA end resection and prevents formation of DNA hairpins","volume":"50","author":"Chen","year":"2013","journal-title":"Mol. Cell"},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1038\/s41594-018-0075-z","article-title":"RPA and RAD51: Fork reversal, fork protection, and genome stability","volume":"25","author":"Bhat","year":"2018","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"369","DOI":"10.1038\/nrm3805","article-title":"Mechanisms and principles of homology search during recombination","volume":"15","author":"Renkawitz","year":"2014","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"749","DOI":"10.1093\/nar\/gkw1125","article-title":"Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament","volume":"45","author":"Ma","year":"2017","journal-title":"Nucleic Acids Res."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"698","DOI":"10.1038\/s41580-019-0152-0","article-title":"DNA double-strand break repair-pathway choice in somatic mammalian cells","volume":"20","author":"Scully","year":"2019","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_72","doi-asserted-by":"crossref","unstructured":"Kowalczykowski, S.C. (2015). An Overview of the Molecular Mechanisms of Recombinational DNA Repair. Cold Spring Harb. Perspect. Biol., 7.","DOI":"10.1101\/cshperspect.a016410"},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"548","DOI":"10.1039\/C7IB00060J","article-title":"Highly efficient genome editing of human hematopoietic stem cells via a nano-silicon-blade delivery approach","volume":"9","author":"Ma","year":"2017","journal-title":"Integr. Biol."},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"464","DOI":"10.1089\/hum.2016.009","article-title":"CRISPRs for Optimal Targeting: Delivery of CRISPR Components as DNA, RNA, and Protein into Cultured Cells and Single-Cell Embryos","volume":"27","author":"Kouranova","year":"2016","journal-title":"Hum. Gene Ther."},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1038\/s41434-022-00344-3","article-title":"Efficient CRISPR\/Cas9 nickase-mediated genome editing in an in vitro model of mucopolysaccharidosis IVA","volume":"30","author":"Leal","year":"2022","journal-title":"Gene Ther."},{"key":"ref_76","doi-asserted-by":"crossref","first-page":"101153","DOI":"10.1016\/j.omtm.2023.101153","article-title":"Iron oxide-coupled CRISPR\/nCas9-based genome editing assessment in mucopolysaccharidosis IVA mice","volume":"31","author":"Leal","year":"2023","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_77","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1186\/s12985-022-01859-2","article-title":"Strategies to improve homology-based repair outcomes following CRISPR-based gene editing in mosquitoes: Lessons in how to keep any repair disruptions local","volume":"19","author":"Finney","year":"2022","journal-title":"Virol. J."},{"key":"ref_78","doi-asserted-by":"crossref","first-page":"985","DOI":"10.1002\/1873-3468.14608","article-title":"Cas9-Geminin and Cdt1-fused anti-CRISPR protein synergistically increase editing accuracy","volume":"597","author":"Matsumoto","year":"2023","journal-title":"FEBS Lett."},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"7355","DOI":"10.1007\/s00253-019-10036-5","article-title":"Recent advances in the engineering and application of streptavidin-like molecules","volume":"103","author":"Le","year":"2019","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"878","DOI":"10.1038\/s41551-017-0145-2","article-title":"Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR-Cas9 genome editing","volume":"1","author":"Paulsen","year":"2017","journal-title":"Nat. Biomed. Eng."},{"key":"ref_81","doi-asserted-by":"crossref","first-page":"3515","DOI":"10.1016\/j.ymthe.2022.09.015","article-title":"AAV vectors: The Rubik\u2019s cube of human gene therapy","volume":"30","author":"Pupo","year":"2022","journal-title":"Mol. Ther."},{"key":"ref_82","doi-asserted-by":"crossref","unstructured":"Zhao, Z., Anselmo, A.C., and Mitragotri, S. (2022). Viral vector-based gene therapies in the clinic. Bioeng. Transl. Med., 7.","DOI":"10.1002\/btm2.10258"},{"key":"ref_83","doi-asserted-by":"crossref","first-page":"e04766","DOI":"10.7554\/eLife.04766","article-title":"Enhanced homology-directed human genome engineering by controlled timing of CRISPR\/Cas9 delivery","volume":"3","author":"Lin","year":"2014","journal-title":"eLife"},{"key":"ref_84","doi-asserted-by":"crossref","first-page":"471","DOI":"10.1038\/s41592-020-0771-6","article-title":"Anti-CRISPR protein applications: Natural brakes for CRISPR-Cas technologies","volume":"17","author":"Marino","year":"2020","journal-title":"Nat. Methods"},{"key":"ref_85","doi-asserted-by":"crossref","unstructured":"Matsumoto, D., Tamamura, H., and Nomura, W. (2020). A cell cycle-dependent CRISPR-Cas9 activation system based on an anti-CRISPR protein shows improved genome editing accuracy. Commun. Biol., 3.","DOI":"10.1038\/s42003-020-01340-2"},{"key":"ref_86","doi-asserted-by":"crossref","first-page":"447","DOI":"10.1089\/crispr.2023.0036","article-title":"Enhancing Precision and Efficiency of Cas9-Mediated Knockin Through Combinatorial Fusions of DNA Repair Proteins","volume":"6","author":"Richardson","year":"2023","journal-title":"CRISPR J."},{"key":"ref_87","doi-asserted-by":"crossref","first-page":"365","DOI":"10.3389\/fgene.2019.00365","article-title":"Enhancement of Precise Gene Editing by the Association of Cas9 With Homologous Recombination Factors","volume":"10","author":"Tran","year":"2019","journal-title":"Front. Genet."},{"key":"ref_88","doi-asserted-by":"crossref","first-page":"182","DOI":"10.1186\/s12934-022-01908-z","article-title":"Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris","volume":"21","author":"Zhang","year":"2022","journal-title":"Microb. Cell Factories"},{"key":"ref_89","doi-asserted-by":"crossref","first-page":"4639","DOI":"10.1093\/nar\/gki778","article-title":"Potentiation of gene targeting in human cells by expression of Saccharomyces cerevisiae Rad52","volume":"33","author":"Galli","year":"2005","journal-title":"Nucleic Acids Res."},{"key":"ref_90","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.biocel.2017.09.012","article-title":"Enhancing CRISPR\/Cas9-mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52","volume":"92","author":"Shao","year":"2017","journal-title":"Int. J. Biochem. Cell Biol."},{"key":"ref_91","doi-asserted-by":"crossref","unstructured":"Carneiro, P., de Freitas, M.V., and Matte, U. (2022). In silico analysis of potential off-target sites to gene editing for Mucopolysaccharidosis type I using the CRISPR\/Cas9 system: Implications for population-specific treatments. PLoS ONE, 17.","DOI":"10.1371\/journal.pone.0262299"},{"key":"ref_92","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1038\/s41596-020-00431-y","article-title":"Tools for experimental and computational analyses of off-target editing by programmable nucleases","volume":"16","author":"Bao","year":"2021","journal-title":"Nat. Protoc."},{"key":"ref_93","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1038\/nbt.3437","article-title":"Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9","volume":"34","author":"Doench","year":"2016","journal-title":"Nat. Biotechnol."},{"key":"ref_94","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1101\/gr.162339.113","article-title":"Analysis of off-target effects of CRISPR\/Cas-derived RNA-guided endonucleases and nickases","volume":"24","author":"Cho","year":"2014","journal-title":"Genome Res."},{"key":"ref_95","doi-asserted-by":"crossref","first-page":"19482","DOI":"10.1038\/s41598-021-98965-y","article-title":"Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair","volume":"11","author":"Schubert","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_96","doi-asserted-by":"crossref","first-page":"4045","DOI":"10.1038\/s41467-019-11962-8","article-title":"Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I","volume":"10","author":"Scharenberg","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_97","doi-asserted-by":"crossref","unstructured":"Wang, Y., Liu, K.I., Sutrisnoh, N.B., Srinivasan, H., Zhang, J., Li, J., Zhang, F., Lalith, C.R.J., Xing, H., and Shanmugam, R. (2018). Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells. Genome Biol., 19.","DOI":"10.1186\/s13059-018-1445-x"},{"key":"ref_98","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1038\/nbt.3481","article-title":"Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA","volume":"34","author":"Richardson","year":"2016","journal-title":"Nat. Biotechnol."},{"key":"ref_99","doi-asserted-by":"crossref","first-page":"2510","DOI":"10.1016\/j.drudis.2022.06.006","article-title":"Small-molecule enhancers of CRISPR-induced homology-directed repair in gene therapy: A medicinal chemist\u2019s perspective","volume":"27","author":"Lee","year":"2022","journal-title":"Drug Discov. Today"},{"key":"ref_100","doi-asserted-by":"crossref","unstructured":"Bischoff, N., Wimberger, S., Maresca, M., and Brakebusch, C. (2020). Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion?. Cells, 9.","DOI":"10.3390\/cells9051318"},{"key":"ref_101","doi-asserted-by":"crossref","first-page":"371","DOI":"10.34172\/bi.2022.23871","article-title":"Advance trends in targeting homology-directed repair for accurate gene editing: An inclusive review of small molecules and modified CRISPR-Cas9 systems","volume":"12","author":"Shams","year":"2022","journal-title":"Bioimpacts"},{"key":"ref_102","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1038\/nbt.4021","article-title":"Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency","volume":"36","author":"Canny","year":"2018","journal-title":"Nat. Biotechnol."},{"key":"ref_103","doi-asserted-by":"crossref","first-page":"2598","DOI":"10.1182\/blood.2020008503","article-title":"Enhanced homology-directed repair for highly efficient gene editing in hematopoietic stem\/progenitor cells","volume":"137","author":"Brault","year":"2021","journal-title":"Blood"},{"key":"ref_104","doi-asserted-by":"crossref","first-page":"2164","DOI":"10.1038\/s41467-018-04609-7","article-title":"Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells","volume":"9","author":"Riesenberg","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_105","doi-asserted-by":"crossref","first-page":"526","DOI":"10.1016\/j.devcel.2018.04.021","article-title":"Tild-CRISPR Allows for Efficient and Precise Gene Knockin in Mouse and Human Cells","volume":"45","author":"Yao","year":"2018","journal-title":"Dev. Cell"},{"key":"ref_106","doi-asserted-by":"crossref","first-page":"801","DOI":"10.1038\/cr.2017.76","article-title":"Homology-mediated end joining-based targeted integration using CRISPR\/Cas9","volume":"27","author":"Yao","year":"2017","journal-title":"Cell Res."},{"key":"ref_107","doi-asserted-by":"crossref","first-page":"969","DOI":"10.1093\/nar\/gkaa1251","article-title":"Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing","volume":"49","author":"Fu","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_108","doi-asserted-by":"crossref","first-page":"e116","DOI":"10.1093\/nar\/gkz669","article-title":"Simultaneous precise editing of multiple genes in human cells","volume":"47","author":"Riesenberg","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_109","doi-asserted-by":"crossref","first-page":"1388","DOI":"10.1038\/s41592-023-01949-1","article-title":"Efficient high-precision homology-directed repair-dependent genome editing by HDRobust","volume":"20","author":"Riesenberg","year":"2023","journal-title":"Nat. Methods"},{"key":"ref_110","doi-asserted-by":"crossref","unstructured":"Hu, Z., Shi, Z., Guo, X., Jiang, B., Wang, G., Luo, D., Chen, Y., and Zhu, Y.S. (2018). Ligase IV inhibitor SCR7 enhances gene editing directed by CRISPR-Cas9 and ssODN in human cancer cells. Cell Biosci., 8.","DOI":"10.1186\/s13578-018-0200-z"},{"key":"ref_111","doi-asserted-by":"crossref","unstructured":"Selvaraj, S., Feist, W.N., Viel, S., Vaidyanathan, S., Dudek, A.M., Gastou, M., Rockwood, S.J., Ekman, F.K., Oseghale, A.R., and Xu, L. (2023). High-efficiency transgene integration by homology-directed repair in human primary cells using DNA-PKcs inhibition. Nat. Biotechnol.","DOI":"10.1038\/s41587-023-01888-4"},{"key":"ref_112","doi-asserted-by":"crossref","unstructured":"Anuchina, A.A., Zaynitdinova, M.I., Demchenko, A.G., Evtushenko, N.A., Lavrov, A.V., and Smirnikhina, S.A. (2023). Bridging Gaps in HDR Improvement: The Role of MAD2L2, SCAI, and SCR7. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24076704"},{"key":"ref_113","doi-asserted-by":"crossref","first-page":"538","DOI":"10.1038\/nbt.3190","article-title":"Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining","volume":"33","author":"Maruyama","year":"2015","journal-title":"Nat. Biotechnol."},{"key":"ref_114","doi-asserted-by":"crossref","unstructured":"Tanihara, F., Hirata, M., Namula, Z., Do, L.T.K., Yoshimura, N., Lin, Q., Takebayashi, K., Sakuma, T., Yamamoto, T., and Otoi, T. (2023). Pigs with an INS point mutation derived from zygotes electroporated with CRISPR\/Cas9 and ssODN. Front. Cell Dev. Biol., 11.","DOI":"10.3389\/fcell.2023.884340"},{"key":"ref_115","doi-asserted-by":"crossref","first-page":"543","DOI":"10.1038\/nbt.3198","article-title":"Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells","volume":"33","author":"Chu","year":"2015","journal-title":"Nat. Biotechnol."},{"key":"ref_116","doi-asserted-by":"crossref","first-page":"e00015-15","DOI":"10.1128\/mSphere.00015-15","article-title":"The Human Adenovirus Type 5 E4orf6\/E1B55K E3 Ubiquitin Ligase Complex Enhances E1A Functional Activity","volume":"1","author":"Dallaire","year":"2016","journal-title":"mSphere"},{"key":"ref_117","doi-asserted-by":"crossref","first-page":"154","DOI":"10.1016\/j.biocel.2018.04.011","article-title":"Suppressing Ku70\/Ku80 expression elevates homology-directed repair efficiency in primary fibroblasts","volume":"99","author":"Li","year":"2018","journal-title":"Int. J. Biochem. Cell Biol."},{"key":"ref_118","doi-asserted-by":"crossref","unstructured":"Li, K., Wang, G., Andersen, T., Zhou, P., and Pu, W.T. (2014). Optimization of genome engineering approaches with the CRISPR\/Cas9 system. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0105779"},{"key":"ref_119","doi-asserted-by":"crossref","first-page":"739","DOI":"10.1016\/j.ygyno.2014.01.034","article-title":"The inhibition of miR-21 promotes apoptosis and chemosensitivity in ovarian cancer","volume":"132","author":"Chan","year":"2014","journal-title":"Gynecol. Oncol."},{"key":"ref_120","doi-asserted-by":"crossref","unstructured":"Jenike, A.E., and Halushka, M.K. (2021). miR-21: A non-specific biomarker of all maladies. Biomark. Res., 9.","DOI":"10.1186\/s40364-021-00272-1"},{"key":"ref_121","doi-asserted-by":"crossref","first-page":"15848","DOI":"10.1073\/pnas.0808046105","article-title":"A chemical compound that stimulates the human homologous recombination protein RAD51","volume":"105","author":"Jayathilaka","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_122","doi-asserted-by":"crossref","first-page":"10548","DOI":"10.1038\/ncomms10548","article-title":"RS-1 enhances CRISPR\/Cas9- and TALEN-mediated knock-in efficiency","volume":"7","author":"Song","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_123","doi-asserted-by":"crossref","first-page":"487","DOI":"10.1038\/nrg.2016.59","article-title":"The molecular hallmarks of epigenetic control","volume":"17","author":"Allis","year":"2016","journal-title":"Nat. Rev. Genet."},{"key":"ref_124","doi-asserted-by":"crossref","unstructured":"Li, G., Zhang, X., Wang, H., Liu, D., Li, Z., Wu, Z., and Yang, H. (2020). Increasing CRISPR\/Cas9-mediated homology-directed DNA repair by histone deacetylase inhibitors. Int. J. Biochem. Cell Biol., 125.","DOI":"10.1016\/j.biocel.2020.105790"},{"key":"ref_125","doi-asserted-by":"crossref","first-page":"1449","DOI":"10.1007\/s11427-020-1855-4","article-title":"HDAC inhibitors improve CRISPR-mediated HDR editing efficiency in iPSCs","volume":"64","author":"Zhang","year":"2021","journal-title":"Sci. China Life Sci."},{"key":"ref_126","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1016\/j.omtn.2022.05.036","article-title":"HDAC inhibitors improve CRISPR-Cas9 mediated prime editing and base editing","volume":"29","author":"Liu","year":"2022","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_127","doi-asserted-by":"crossref","first-page":"473","DOI":"10.1089\/crispr.2023.0033","article-title":"Trichostatin A for Efficient CRISPR-Cas9 Gene Editing of Human Pluripotent Stem Cells","volume":"6","author":"Molugu","year":"2023","journal-title":"CRISPR J."},{"key":"ref_128","doi-asserted-by":"crossref","first-page":"e2103630118","DOI":"10.1073\/pnas.2103630118","article-title":"Ku70 suppresses alternative end joining in G1-arrested progenitor B cells","volume":"118","author":"Liang","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_129","doi-asserted-by":"crossref","first-page":"653","DOI":"10.1098\/rstb.2008.0196","article-title":"Non-homologous end joining in class switch recombination: The beginning of the end","volume":"364","author":"Kotnis","year":"2009","journal-title":"Philos. Trans. R. Soc. Lond. B Biol. Sci."},{"key":"ref_130","doi-asserted-by":"crossref","unstructured":"Sishc, B.J., and Davis, A.J. (2017). The Role of the Core Non-Homologous End Joining Factors in Carcinogenesis and Cancer. Cancers, 9.","DOI":"10.3390\/cancers9070081"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/5\/2456\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:02:31Z","timestamp":1760104951000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/5\/2456"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,2,20]]},"references-count":130,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2024,3]]}},"alternative-id":["ijms25052456"],"URL":"https:\/\/doi.org\/10.3390\/ijms25052456","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,2,20]]}}}