{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T01:36:49Z","timestamp":1760060209603,"version":"build-2065373602"},"reference-count":37,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2025,8,18]],"date-time":"2025-08-18T00:00:00Z","timestamp":1755475200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"The Ministry of Science and Higher Education of the Russian Federation for RCMG"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Prime editing is a promising approach for correcting pathogenic variants, but its efficiency remains variable across genomic contexts. Here, we systematically evaluated 12 modifications of the PEmax system for correcting the CFTR F508del pathogenic variant that caused cystic fibrosis in patient-derived airway basal cells. We chose EXO1 and FEN1 nucleases to improve the original system. While all tested variants showed comparatively low efficiency in this AT-rich genomic region, 4-FEN modification demonstrated significantly improved editing rates (up to 2.13 fold) compared to standard PEmax. Our results highlight two key findings: first, the persistent challenge of AT-rich target sequence correction even with optimized editors, and second, the performance of 4-FEN suggests its potential value for other genomic targets.<\/jats:p>","DOI":"10.3390\/ijms26167943","type":"journal-article","created":{"date-parts":[[2025,8,18]],"date-time":"2025-08-18T16:22:33Z","timestamp":1755534153000},"page":"7943","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Prime Editing Modification with FEN1 Improves F508del Variant Editing in the CFTR Gene in Airway Basal Cells"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7086-2288","authenticated-orcid":false,"given":"Olga V.","family":"Volodina","sequence":"first","affiliation":[{"name":"Laboratory of Genome Editing, Research Centre for Medical Genetics, 115522 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4460-7627","authenticated-orcid":false,"given":"Anna G.","family":"Demchenko","sequence":"additional","affiliation":[{"name":"Laboratory of Genome Editing, Research Centre for Medical Genetics, 115522 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1820-5461","authenticated-orcid":false,"given":"Arina A.","family":"Anuchina","sequence":"additional","affiliation":[{"name":"Laboratory of Genome Editing, Research Centre for Medical Genetics, 115522 Moscow, Russia"},{"name":"Laboratory for Structural Analysis and Engineering of Membrane Systems, The Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 141701 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1285-9093","authenticated-orcid":false,"given":"Oxana P.","family":"Ryzhkova","sequence":"additional","affiliation":[{"name":"The Shared Resource Centre \u201cGenome\u201d, Research Centre for Medical Genetics, 115522 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8728-8574","authenticated-orcid":false,"given":"Valeriia A.","family":"Kovalskaya","sequence":"additional","affiliation":[{"name":"The Shared Resource Centre \u201cGenome\u201d, Research Centre for Medical Genetics, 115522 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0770-2569","authenticated-orcid":false,"given":"Ekaterina V.","family":"Kondrateva","sequence":"additional","affiliation":[{"name":"Laboratory of Genome Editing, Research Centre for Medical Genetics, 115522 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-0997-9257","authenticated-orcid":false,"given":"Ekaterina V.","family":"Artemova","sequence":"additional","affiliation":[{"name":"Laboratory of Genome Editing, Research Centre for Medical Genetics, 115522 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0291-153X","authenticated-orcid":false,"given":"Vyacheslav Y.","family":"Tabakov","sequence":"additional","affiliation":[{"name":"The Shared Resource Centre \u201cBiobank\u201d, Research Centre for Medical Genetics, 115522 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0381-5267","authenticated-orcid":false,"given":"Maxim A.","family":"Ignatov","sequence":"additional","affiliation":[{"name":"Laboratory of Radiation Biophysics, Department of Experimental Radiobiology and Radiation Medicine, State Research Center-Burnasyan Federal Medical Biophysical Center of Federal Medical Biological Agency (SRC-FMBC), 123098 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9008-4401","authenticated-orcid":false,"given":"Natalia Y.","family":"Vorobyeva","sequence":"additional","affiliation":[{"name":"Laboratory of Radiation Biophysics, Department of Experimental Radiobiology and Radiation Medicine, State Research Center-Burnasyan Federal Medical Biophysical Center of Federal Medical Biological Agency (SRC-FMBC), 123098 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5921-9056","authenticated-orcid":false,"given":"Andreyan N.","family":"Osipov","sequence":"additional","affiliation":[{"name":"Laboratory of Radiation Biophysics, Department of Experimental Radiobiology and Radiation Medicine, State Research Center-Burnasyan Federal Medical Biophysical Center of Federal Medical Biological Agency (SRC-FMBC), 123098 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4962-6947","authenticated-orcid":false,"given":"Alexander V.","family":"Lavrov","sequence":"additional","affiliation":[{"name":"Laboratory of Genome Editing, Research Centre for Medical Genetics, 115522 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1558-3048","authenticated-orcid":false,"given":"Svetlana A.","family":"Smirnikhina","sequence":"additional","affiliation":[{"name":"Laboratory of Genome Editing, Research Centre for Medical Genetics, 115522 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"4258","DOI":"10.1038\/s41467-020-18101-8","article-title":"CFTR trafficking mutations disrupt cotranslational protein folding by targeting biosynthetic intermediates","volume":"11","author":"Shishido","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"575","DOI":"10.1002\/humu.10041","article-title":"Cystic fibrosis: A worldwide analysis of CFTR mutations\u2014Correlation with incidence data and application to screening","volume":"19","author":"Bobadilla","year":"2002","journal-title":"Hum. Mutat."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Petrova, N., Balinova, N., Marakhonov, A., Vasilyeva, T., Kashirskaya, N., Galkina, V., Ginter, E., Kutsev, S., and Zinchenko, R. (2021). Ethnic Differences in the Frequency of CFTR Gene Mutations in Populations of the European and North Caucasian Part of the Russian Federation. Front. Genet., 12.","DOI":"10.3389\/fgene.2021.678374"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"893","DOI":"10.1111\/apa.15155","article-title":"Cystic fibrosis in the year 2020: A disease with a new face","volume":"109","year":"2020","journal-title":"Acta Paediatr."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1061","DOI":"10.1016\/j.jcf.2022.05.001","article-title":"Dose adjustments of Elexacaftor\/Tezacaftor\/Ivacaftor in response to mental health side effects in adults with cystic fibrosis","volume":"21","author":"Spoletini","year":"2022","journal-title":"J. Cyst. Fibros."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e27382","DOI":"10.1002\/ppul.27382","article-title":"Heterogeneity in Reported Side Effects Following Initiation of Elexacaftor-Tezacaftor-Ivacaftor: Experiences at a Quaternary CF Care Center","volume":"60","author":"Papadakis","year":"2024","journal-title":"Pediatr. Pulmonol."},{"key":"ref_7","unstructured":"(2025, March 10). Safety and Possible Side Effects|TRIKAFTA\u00ae (Elexacaftor\/Tezacaftor\/Ivacaftor and Ivacaftor). (n.d.). Available online: https:\/\/www.trikafta.com\/safety-side-effects."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"5635","DOI":"10.1016\/j.cell.2021.09.018","article-title":"Enhanced prime editing systems by manipulating cellular determinants of editing outcomes","volume":"184","author":"Chen","year":"2021","journal-title":"Cell"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"396","DOI":"10.1038\/s41434-021-00263-9","article-title":"Prime editing\u2014An update on the field","volume":"28","author":"Scholefield","year":"2021","journal-title":"Gene Ther."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1038\/s41576-022-00541-1","article-title":"Prime editing for precise and highly versatile genome manipulation","volume":"24","author":"Chen","year":"2022","journal-title":"Nat. Rev. Genet."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1038\/s41587-021-01039-7","article-title":"Engineered pegRNAs improve prime editing efficiency","volume":"40","author":"Nelson","year":"2021","journal-title":"Nat. Biotechnol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Westberg, I., Carlsen, F.M., Johansen, I.E., and Petersen, B.L. (2023). Cytosine base editors optimized for genome editing in potato protoplasts. Front. Genome Ed., 5.","DOI":"10.3389\/fgeed.2023.1247702"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4793","DOI":"10.1093\/nar\/gkab211","article-title":"Rational engineering of a modular bacterial CRISPR\u2013Cas activation platform with expanded target range","volume":"49","author":"Kcam","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_14","unstructured":"(2025, July 03). FEN1 Flap Structure-Specific Endonuclease 1 [Homo Sapiens (Human)]\u2014Gene\u2014NCBI. (n.d.), Available online: https:\/\/www.ncbi.nlm.nih.gov\/gene\/2237."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Keijzers, G., Bohr, V.A., and Rasmussen, L.J. (2015). Human exonuclease 1 (EXO1) activity characterization and its function on flap structures. Biosci. Rep., 35.","DOI":"10.1042\/BSR20150058"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"101544","DOI":"10.1016\/j.xcrm.2024.101544","article-title":"Prime editing functionally corrects cystic fibrosis-causing CFTR mutations in human organoids and airway epithelial cells","volume":"5","author":"Bulcaen","year":"2024","journal-title":"Cell Rep. Med."},{"key":"ref_17","unstructured":"(2025, July 03). Selection of Optimal pegRNAs to Enhance Efficiency of Prime Editing in AT-Rich Genome Regions|Biochemistry (Moscow). (n.d.). Available online: https:\/\/link.springer.com\/article\/10.1134\/S0006297924604672#citeas."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Ma, X., Shao, Y., Tian, L., Flasch, D.A., Mulder, H.L., Edmonson, M.N., Liu, Y., Chen, X., Newman, S., and Nakitandwe, J. (2019). Analysis of error profiles in deep next-generation sequencing data. Genome Biol., 20.","DOI":"10.1186\/s13059-019-1659-6"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"5634","DOI":"10.1093\/nar\/gky313","article-title":"Flap endonuclease overexpression drives genome instability and DNA damage hypersensitivity in a PCNA-dependent manner","volume":"46","author":"Becker","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"15317","DOI":"10.1002\/cam4.6188","article-title":"Androgen receptor knockdown enhances prostate cancer chemosensitivity by down-regulating FEN1 through the ERK\/ELK1 signalling pathway","volume":"12","author":"Xie","year":"2023","journal-title":"Cancer Med."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1186\/s12967-025-06216-9","article-title":"Inhibition of the FEN1-PBX1 axis elicits cellular senescence in breast cancer via the increased intracellular reactive oxygen species levels","volume":"23","author":"Wu","year":"2025","journal-title":"J. Transl. Med."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Berfelde, J., Hildebrand, L.S., Kuhlmann, L., Fietkau, R., and Distel, L.V. (2024). FEN1 Inhibition as a Potential Novel Targeted Therapy against Breast Cancer and the Prognostic Relevance of FEN1. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25042110"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2386","DOI":"10.1080\/15384101.2018.1534511","article-title":"EXO1 overexpression is associated with poor prognosis of hepatocellular carcinoma patients","volume":"17","author":"Dai","year":"2018","journal-title":"Cell Cycle"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"3983","DOI":"10.1016\/j.cell.2023.07.039","article-title":"Phage-assisted evolution and protein engineering yield compact, efficient prime editors","volume":"186","author":"Doman","year":"2023","journal-title":"Cell"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1038\/s41586-024-07259-6","article-title":"Improving prime editing with an endogenous small RNA-binding protein","volume":"628","author":"Yan","year":"2024","journal-title":"Nature"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"5617","DOI":"10.1038\/s41467-021-25928-2","article-title":"Generation of a more efficient prime editor 2 by addition of the Rad51 DNA-binding domain","volume":"12","author":"Song","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Park, S.-J., Jeong, T.Y., Shin, S.K., Yoon, D.E., Lim, S.-Y., Kim, S.P., Choi, J., Lee, H., Hong, J.-I., and Ahn, J. (2021). Targeted mutagenesis in mouse cells and embryos using an enhanced prime editor. Genome Biol., 22.","DOI":"10.1186\/s13059-021-02389-w"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"3512","DOI":"10.1038\/s41467-022-31270-y","article-title":"Peptide fusion improves prime editing efficiency","volume":"13","author":"Velimirovic","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1038\/s41592-023-02162-w","article-title":"Exonuclease-enhanced prime editors","volume":"21","author":"Truong","year":"2024","journal-title":"Nat. Methods"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1038\/ng0992-21","article-title":"Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis","volume":"2","author":"Johnson","year":"1992","journal-title":"Nat. Genet."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Pfeiffer, F., Gr\u00f6ber, C., Blank, M., H\u00e4ndler, K., Beyer, M., Schultze, J.L., and Mayer, G. (2018). Systematic evaluation of error rates and causes in short samples in next-generation sequencing. Sci. Rep., 8.","DOI":"10.1038\/s41598-018-29325-6"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"101933","DOI":"10.1016\/j.scr.2020.101933","article-title":"Generation of induced pluripotent stem cell line (RCMGi001-A) from human skin fibroblasts of a cystic fibrosis patient with p.F508del mutation","volume":"48","author":"Kondrateva","year":"2020","journal-title":"Stem Cell Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"102896","DOI":"10.1016\/j.scr.2022.102896","article-title":"Generation of two induced pluripotent stem cell lines (RCMGi005-A\/B) from human skin fibroblasts of a cystic fibrosis patient with homozygous F508del mutation in CFTR gene","volume":"64","author":"Panchuk","year":"2022","journal-title":"Stem Cell Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"102251","DOI":"10.1016\/j.scr.2021.102251","article-title":"Derivation of iPSC line (RCMGi002-A) from dermal fibroblasts of a cystic fibrosis female patient with homozygous F508del mutation","volume":"53","author":"Kondrateva","year":"2021","journal-title":"Stem Cell Res."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Demchenko, A., Belova, L., Balyasin, M., Kochergin-Nikitsky, K., Kondrateva, E., Voronina, E., Pozhitnova, V., Tabakov, V., Salikhova, D., and Bukharova, T. (2024). Airway basal cells from human-induced pluripotent stem cells: A new frontier in cystic fibrosis research. Front. Cell Dev. Biol., 12.","DOI":"10.3389\/fcell.2024.1336392"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1038\/s41587-019-0032-3","article-title":"CRISPResso2 provides accurate and rapid genome editing sequence analysis","volume":"37","author":"Clement","year":"2019","journal-title":"Nat. Biotechnol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1473","DOI":"10.1093\/bioinformatics\/btu048","article-title":"Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases","volume":"30","author":"Bae","year":"2014","journal-title":"Bioinformatics"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/16\/7943\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:29:35Z","timestamp":1760034575000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/16\/7943"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,18]]},"references-count":37,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2025,8]]}},"alternative-id":["ijms26167943"],"URL":"https:\/\/doi.org\/10.3390\/ijms26167943","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2025,8,18]]}}}