{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T20:28:38Z","timestamp":1785788918889,"version":"3.56.0"},"reference-count":14,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T00:00:00Z","timestamp":1782259200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000900","name":"California Institute for Regenerative Medicine","doi-asserted-by":"publisher","award":["TRAN1-09292"],"award-info":[{"award-number":["TRAN1-09292"]}],"id":[{"id":"10.13039\/100000900","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000900","name":"California Institute for Regenerative Medicine","doi-asserted-by":"publisher","award":["CLIN1-11497"],"award-info":[{"award-number":["CLIN1-11497"]}],"id":[{"id":"10.13039\/100000900","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000900","name":"California Institute for Regenerative Medicine","doi-asserted-by":"publisher","award":["CLIN2-11722"],"award-info":[{"award-number":["CLIN2-11722"]}],"id":[{"id":"10.13039\/100000900","id-type":"DOI","asserted-by":"publisher"}]},{"award":["TRAN1-09292"],"award-info":[{"award-number":["TRAN1-09292"]}],"id":[{"id":"https:\/\/ror.org\/033m8b439","id-type":"ROR","asserted-by":"publisher"}]},{"award":["CLIN1-11497"],"award-info":[{"award-number":["CLIN1-11497"]}],"id":[{"id":"https:\/\/ror.org\/033m8b439","id-type":"ROR","asserted-by":"publisher"}]},{"award":["CLIN2-11722"],"award-info":[{"award-number":["CLIN2-11722"]}],"id":[{"id":"https:\/\/ror.org\/033m8b439","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000050","name":"NIH Cure Sickle Cell\/NHLBI","doi-asserted-by":"publisher","award":["OT2 HL151319"],"award-info":[{"award-number":["OT2 HL151319"]}],"id":[{"id":"10.13039\/100000050","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000050","name":"NIH Cure Sickle Cell\/NHLBI","doi-asserted-by":"publisher","award":["OT3HL147741"],"award-info":[{"award-number":["OT3HL147741"]}],"id":[{"id":"10.13039\/100000050","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>Background\/Objectives: Single-stranded oligonucleotides (ssODNs) are used as donor templates for therapeutic gene editing by CRISPR-Cas9 cleavage and homology-directed repair (HDR). Although ssODN sequence fidelity is critical to the safety and efficacy of editing, standard quality control methods cannot resolve individual nucleotide errors. Methods: We performed deep sequencing of ssODNs from three manufacturers and amplicons from edited hematopoietic stem\/progenitor cells. Results: We find that synthesis errors are present in all ssODNs tested at rates that vary more than two-fold among manufacturers, at positions that are dependent on sequence context. These synthesis errors are propagated into the genome by HDR at frequencies proportional to their abundance in the ssODN. In our sickle cell mutation correction protocol, the most prevalent SNEs are predicted to produce benign \u03b2-globin variants, while the less frequent frameshift deletions are predicted to generate \u03b2-thalassemia-like alleles. Conclusions: Current quality control standards are insufficient to detect these errors, and deep sequencing of ssODNs should be incorporated into regulatory submissions for clinical gene editing programs.<\/jats:p>","DOI":"10.3390\/genes17070729","type":"journal-article","created":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T14:27:52Z","timestamp":1783434472000},"page":"729","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Oligonucleotide Synthesis Errors Are a Source of Untoward Variation in HDR-Mediated Gene Editing"],"prefix":"10.3390","volume":"17","author":[{"given":"Stacia K.","family":"Wyman","sequence":"first","affiliation":[{"name":"Innovative Genomics Institute, University of California, Berkeley, CA 94720, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8305-8184","authenticated-orcid":false,"given":"Zulema","family":"Romero","sequence":"additional","affiliation":[{"name":"Departments of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-4830-8319","authenticated-orcid":false,"given":"Seok-Jin","family":"Heo","sequence":"additional","affiliation":[{"name":"Laboratory for Genomics Research, Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marian","family":"Navarrete","sequence":"additional","affiliation":[{"name":"Departments of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Netravathi","family":"Krishnappa","sequence":"additional","affiliation":[{"name":"Innovative Genomics Institute, University of California, Berkeley, CA 94720, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1840-6087","authenticated-orcid":false,"given":"Donald B.","family":"Kohn","sequence":"additional","affiliation":[{"name":"Departments of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David I. K.","family":"Martin","sequence":"additional","affiliation":[{"name":"UCSF Benioff Children\u2019s Hospital Oakland, Oakland, CA 94609, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark C.","family":"Walters","sequence":"additional","affiliation":[{"name":"UCSF Benioff Children\u2019s Hospital Oakland, Oakland, CA 94609, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dario","family":"Boffelli","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Cedars-Sinai Guerin Children\u2019s, Los Angeles, CA 90069, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Pruett-Miller, S.M. (2015). Gene Editing Using ssODNs with Engineered Endonucleases. Chromosomal Mutagenesis, Springer.","DOI":"10.1007\/978-1-4939-1862-1"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1384","DOI":"10.1093\/clinchem\/hvab136","article-title":"Digital Quantification of Chemical Oligonucleotide Synthesis Errors","volume":"67","author":"Filges","year":"2021","journal-title":"Clin. Chem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"12095","DOI":"10.1038\/s41598-022-16222-2","article-title":"Quantification of Synthetic Errors during Chemical Synthesis of DNA and Its Suppression by Non-Canonical Nucleosides","volume":"12","author":"Masaki","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Yakovleva, K.I., Pereverzev, I.M., Kechin, A.A., Boyarskikh, U.A., Filipenko, M.L., Shevelev, G.Y., Sherstyuk, Y.V., and Dovydenko, I.S. (2025). The Influence of the Capping Step During Solid-Phase Phosphoramidite Synthesis of Oligonucleotides on Synthetic Errors in Oligonucleotides. Molecules, 31.","DOI":"10.3390\/molecules31010094"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.omtn.2017.02.006","article-title":"Optimizing the DNA Donor Template for Homology-Directed Repair of Double-Strand Breaks","volume":"7","author":"Song","year":"2017","journal-title":"Mol. Ther.-Nucleic Acids"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"969","DOI":"10.1093\/nar\/gkaa1251","article-title":"Dynamics and Competition of CRISPR\u2013Cas9 Ribonucleoproteins and AAV Donor-Mediated NHEJ, MMEJ and HDR Editing","volume":"49","author":"Fu","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"360ra134","DOI":"10.1126\/scitranslmed.aaf9336","article-title":"Selection-Free Genome Editing of the Sickle Mutation in Human Adult Hematopoietic Stem\/Progenitor Cells","volume":"8","author":"DeWitt","year":"2016","journal-title":"Sci. Transl. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"104374","DOI":"10.1016\/j.isci.2022.104374","article-title":"High-Level Correction of the Sickle Mutation Is Amplified in Vivo during Erythroid Differentiation","volume":"25","author":"Magis","year":"2022","journal-title":"iScience"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"4321","DOI":"10.1182\/blood-2025-4321","article-title":"Synthetic Oligonucleotides Contain Synthesis Errors That Are Propagated into the Genome by Editing through Homology-Directed Repair","volume":"146","author":"Wyman","year":"2025","journal-title":"Blood"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"595","DOI":"10.2169\/internalmedicine.42.595","article-title":"Two Cases in Which an Abnormal Hemoglobin (Hb Kamakura) Was Identified on the Basis of an Abnormally Low Glycohemoglobin Levels","volume":"42","author":"Saisho","year":"2003","journal-title":"Intern. 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Blood Cancer"}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/17\/7\/729\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,8]],"date-time":"2026-07-08T13:06:18Z","timestamp":1783515978000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/17\/7\/729"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,24]]},"references-count":14,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["genes17070729"],"URL":"https:\/\/doi.org\/10.3390\/genes17070729","relation":{},"ISSN":["2073-4425"],"issn-type":[{"value":"2073-4425","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,24]]}}}