{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T01:33:23Z","timestamp":1790732003461,"version":"4.1.0"},"reference-count":52,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2022,5,25]],"date-time":"2022-05-25T00:00:00Z","timestamp":1653436800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003399","name":"Science and Technology Commission of Shanghai Municipality","doi-asserted-by":"publisher","award":["19YF1403500"],"award-info":[{"award-number":["19YF1403500"]}],"id":[{"id":"10.13039\/501100003399","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003399","name":"Science and Technology Commission of Shanghai Municipality","doi-asserted-by":"publisher","award":["2019M651353"],"award-info":[{"award-number":["2019M651353"]}],"id":[{"id":"10.13039\/501100003399","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003399","name":"Science and Technology Commission of Shanghai Municipality","doi-asserted-by":"publisher","award":["cstc2020jcyj-zdxm 0180"],"award-info":[{"award-number":["cstc2020jcyj-zdxm 0180"]}],"id":[{"id":"10.13039\/501100003399","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003399","name":"Science and Technology Commission of Shanghai Municipality","doi-asserted-by":"publisher","award":["cstc2019jxjl130001"],"award-info":[{"award-number":["cstc2019jxjl130001"]}],"id":[{"id":"10.13039\/501100003399","id-type":"DOI","asserted-by":"publisher"}]},{"name":"China Postdoctoral Science Foundation funded project","award":["19YF1403500"],"award-info":[{"award-number":["19YF1403500"]}]},{"name":"China Postdoctoral Science Foundation funded project","award":["2019M651353"],"award-info":[{"award-number":["2019M651353"]}]},{"name":"China Postdoctoral Science Foundation funded project","award":["cstc2020jcyj-zdxm 0180"],"award-info":[{"award-number":["cstc2020jcyj-zdxm 0180"]}]},{"name":"China Postdoctoral Science Foundation funded project","award":["cstc2019jxjl130001"],"award-info":[{"award-number":["cstc2019jxjl130001"]}]},{"name":"Chongqing Natural Science Foundation","award":["19YF1403500"],"award-info":[{"award-number":["19YF1403500"]}]},{"name":"Chongqing Natural Science Foundation","award":["2019M651353"],"award-info":[{"award-number":["2019M651353"]}]},{"name":"Chongqing Natural Science Foundation","award":["cstc2020jcyj-zdxm 0180"],"award-info":[{"award-number":["cstc2020jcyj-zdxm 0180"]}]},{"name":"Chongqing Natural Science Foundation","award":["cstc2019jxjl130001"],"award-info":[{"award-number":["cstc2019jxjl130001"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Prime editing was used to insert and correct various pathogenic mutations except for beta-thalassemia variants, which disrupt functional beta-globin and prevent hemoglobin assembly in erythrocytes. This study investigated the effect of gene correction using prime editor version 3 (PE3) in a mouse model with the human beta-thalassemia IVS-II-654 mutation (C &gt; T). The T conversion generates a 5\u2032 donor site at intron 2 of the beta-globin gene resulting in aberrant splicing of pre-mRNA, which affects beta-globin expression. We microinjected PE3 components (pegRNA, nick sgRNA, and PE2 mRNA) into the zygotes from IVS-II-654 mice to generate mutation-edited mice. Genome sequencing of the IVS-II-654 site showed that PE3 installed the correction (T &gt; C), with an editing efficiency of 14.29%. Reverse transcription-PCR analysis showed that the PE3-induced conversion restored normal splicing of beta-globin mRNA. Subsequent comprehensive phenotypic analysis of thalassemia symptoms, including anemic hematological parameters, anisocytosis, splenomegaly, cardiac hypertrophy, extramedullary hematopoiesis, and iron overload, showed that the corrected IVS-II-654 mice had a normal phenotype identical to the wild type mice. Off-target analysis of pegRNA and nick sgRNA additionally showed the genomic safety of PE3. These results suggest that correction of beta-thalassemia mutation by PE3 may be a straightforward therapeutic strategy for this disease.<\/jats:p>","DOI":"10.3390\/ijms23115948","type":"journal-article","created":{"date-parts":[[2022,5,26]],"date-time":"2022-05-26T08:50:22Z","timestamp":1653555022000},"page":"5948","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":28,"title":["Correction of Beta-Thalassemia IVS-II-654 Mutation in a Mouse Model Using Prime Editing"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2466-949X","authenticated-orcid":false,"given":"Haokun","family":"Zhang","sequence":"first","affiliation":[{"name":"State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, School of Life Sciences, Fudan University, Shanghai 200438, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruilin","family":"Sun","sequence":"additional","affiliation":[{"name":"Shanghai Model Organisms Center, No.3577 Jinke Rd., Shanghai 201203, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jian","family":"Fei","sequence":"additional","affiliation":[{"name":"Shanghai Model Organisms Center, No.3577 Jinke Rd., Shanghai 201203, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongyan","family":"Chen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, School of Life Sciences, Fudan University, Shanghai 200438, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daru","family":"Lu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, School of Life Sciences, Fudan University, Shanghai 200438, China"},{"name":"NHC Key Laboratory of Birth Defects and Reproductive Health, Chongqing Key Laboratory of Birth Defects and Reproductive Health, Chongqing Population and Family Planning, Science and Technology Research Institute, Chongqing 404100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,5,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1016\/j.bcmd.2017.06.001","article-title":"Molecular basis of beta thalassemia and potential therapeutic targets","volume":"70","author":"Thein","year":"2018","journal-title":"Blood Cells Mol. Dis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"480","DOI":"10.2471\/BLT.06.036673","article-title":"Global epidemiology of haemoglobin disorders and derived service indicators","volume":"86","author":"Modell","year":"2008","journal-title":"Bull. World Health Organ."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"113","DOI":"10.3109\/03630269809092136","article-title":"Access to a syllabus of human hemoglobin variants (1996) via the World Wide Web","volume":"22","author":"Hardison","year":"1998","journal-title":"Hemoglobin"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"541","DOI":"10.1111\/j.1365-2141.1994.tb05071.x","article-title":"RNA transcripts of the beta-thalassaemia allele IVS-2-654 C-->T: A small amount of normally processed beta-globin mRNA is still produced from the mutant gene","volume":"88","author":"Huang","year":"1994","journal-title":"Br. J. Haematol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2152","DOI":"10.1182\/blood.V91.6.2152","article-title":"A common human beta globin splicing mutation modeled in mice","volume":"91","author":"Lewis","year":"1998","journal-title":"Blood"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e1788","DOI":"10.1002\/mgg3.1788","article-title":"Current status of beta-thalassemia and its treatment strategies","volume":"9","author":"Ali","year":"2021","journal-title":"Mol. Genet. Genom. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1089\/hum.2017.228","article-title":"Recent Progress on Genetic Diagnosis and Therapy for beta-Thalassemia in China and Around the World","volume":"29","author":"Zhang","year":"2018","journal-title":"Hum. Gene Ther."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2616","DOI":"10.1093\/hmg\/ddm218","article-title":"Restoration of the balanced alpha\/beta-globin gene expression in beta654-thalassemia mice using combined RNAi and antisense RNA approach","volume":"16","author":"Xie","year":"2007","journal-title":"Hum. Mol. Genet."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"595","DOI":"10.1016\/S1673-8527(08)60080-6","article-title":"Amelioration of beta654-thalassemia in mouse model with the knockdown of aberrantly spliced beta-globin mRNA","volume":"35","author":"Xie","year":"2008","journal-title":"J. Genet. Genom."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1007\/s12185-010-0727-1","article-title":"Correction of beta654-thalassaemia mice using direct intravenous injection of siRNA and antisense RNA vectors","volume":"93","author":"Xie","year":"2011","journal-title":"Int. J. Hematol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"356","DOI":"10.3324\/haematol.12010","article-title":"A novel transgenic mouse model produced from lentiviral germline integration for the study of beta-thalassemia gene therapy","volume":"93","author":"Li","year":"2008","journal-title":"Haematologica"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1304","DOI":"10.3324\/haematol.2013.087916","article-title":"Therapeutic effects of induced pluripotent stem cells in chimeric mice with beta-thalassemia","volume":"99","author":"Yang","year":"2014","journal-title":"Haematologica"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e12491","DOI":"10.1111\/cpr.12491","article-title":"Treatment of beta(654)-thalassaemia by TALENs in a mouse model","volume":"51","author":"Fang","year":"2018","journal-title":"Cell Prolif."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Lu, D., Gong, X., Fang, Y., Guo, X., Chen, Y., Yang, F., Zhao, G., Ma, Q., Zeng, Y., and Zeng, F. (2021). Correction of RNA splicing defect in beta(654)-thalassemia mice using CRISPR\/Cas9 gene-editing technology. Haematologica.","DOI":"10.3324\/haematol.2020.278238"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1038\/s41586-019-1711-4","article-title":"Search-and-replace genome editing without double-strand breaks or donor DNA","volume":"576","author":"Anzalone","year":"2019","journal-title":"Nature"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"618406","DOI":"10.3389\/fgeed.2021.618406","article-title":"Base and Prime Editing Technologies for Blood Disorders","volume":"3","author":"Antoniou","year":"2021","journal-title":"Front. Genome Ed."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1038\/s41587-021-00901-y","article-title":"CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells","volume":"40","author":"Petri","year":"2022","journal-title":"Nat. Biotechnol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"824","DOI":"10.1038\/s41587-020-0561-9","article-title":"Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors","volume":"38","author":"Anzalone","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1016\/j.ymeth.2021.03.013","article-title":"Progression and application of CRISPR-Cas genomic editors","volume":"194","author":"Yang","year":"2021","journal-title":"Methods"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Koniali, L., Lederer, C.W., and Kleanthous, M. (2021). Therapy Development by Genome Editing of Hematopoietic Stem Cells. Cells, 10.","DOI":"10.3390\/cells10061492"},{"key":"ref_21","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_22","doi-asserted-by":"crossref","first-page":"354","DOI":"10.1182\/asheducation-2013.1.354","article-title":"Genetic association studies in beta-hemoglobinopathies","volume":"2013","author":"Thein","year":"2013","journal-title":"Hematol. Am. Soc. Hematol. Educ. Program."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"358","DOI":"10.1038\/nprot.2017.143","article-title":"CRISPR\/Cas9 genome editing in human hematopoietic stem cells","volume":"13","author":"Bak","year":"2018","journal-title":"Nat. Protoc."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Malik, P., and Tisdale, J. (2017). Gene and Cell Therapies for Beta-Globinopathies. American Society of Gene Cell Therapy, Springer. [1st ed.].","DOI":"10.1007\/978-1-4939-7299-9"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"420","DOI":"10.1038\/nature17946","article-title":"Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage","volume":"533","author":"Komor","year":"2016","journal-title":"Nature"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"21","DOI":"10.3389\/fgene.2018.00021","article-title":"Commentary: Programmable base editing of A.T to G.C in genomic DNA without DNA cleavage","volume":"9","author":"Matsoukas","year":"2018","journal-title":"Front. Genet."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1839","DOI":"10.1126\/science.1165409","article-title":"Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A","volume":"322","author":"Sankaran","year":"2008","journal-title":"Science"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1093","DOI":"10.1038\/nature08243","article-title":"Developmental and species-divergent globin switching are driven by BCL11A","volume":"460","author":"Sankaran","year":"2009","journal-title":"Nature"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"140","DOI":"10.1016\/j.bcmd.2010.05.006","article-title":"Binding patterns of BCL11A in the globin and GATA1 loci and characterization of the BCL11A fetal hemoglobin locus","volume":"45","author":"Jawaid","year":"2010","journal-title":"Blood Cells Mol. Dis."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"742","DOI":"10.1038\/ng.637","article-title":"KLF1 regulates BCL11A expression and gamma- to beta-globin gene switching","volume":"42","author":"Zhou","year":"2010","journal-title":"Nat. Genet."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"6518","DOI":"10.1073\/pnas.1303976110","article-title":"Corepressor-dependent silencing of fetal hemoglobin expression by BCL11A","volume":"110","author":"Xu","year":"2013","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1038\/nature15521","article-title":"BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis","volume":"527","author":"Canver","year":"2015","journal-title":"Nature"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"776","DOI":"10.1038\/s41591-019-0401-y","article-title":"Highly efficient therapeutic gene editing of human hematopoietic stem cells","volume":"25","author":"Wu","year":"2019","journal-title":"Nat. Med."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1038\/s41591-020-0790-y","article-title":"Therapeutic base editing of human hematopoietic stem cells","volume":"26","author":"Zeng","year":"2020","journal-title":"Nat. Med."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"6677","DOI":"10.1172\/JCI140189","article-title":"BCL11A enhancer-edited hematopoietic stem cells persist in rhesus monkeys without toxicity","volume":"130","author":"Demirci","year":"2020","journal-title":"J. Clin. Invest."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2370","DOI":"10.1111\/pbi.13399","article-title":"Engineering herbicide resistance via prime editing in rice","volume":"18","author":"Butt","year":"2020","journal-title":"Plant. Biotechnol. J."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"582","DOI":"10.1038\/s41587-020-0455-x","article-title":"Prime genome editing in rice and wheat","volume":"38","author":"Lin","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"667","DOI":"10.1016\/j.molp.2020.03.010","article-title":"Plant Prime Editors Enable Precise Gene Editing in Rice Cells","volume":"13","author":"Tang","year":"2020","journal-title":"Mol. Plant."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2001","DOI":"10.1016\/j.ymthe.2021.02.022","article-title":"Engineered prime editors with PAM flexibility","volume":"29","author":"Kweon","year":"2021","journal-title":"Mol. Ther."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2121","DOI":"10.1038\/s41467-021-22295-w","article-title":"Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice","volume":"12","author":"Liu","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"W242","DOI":"10.1093\/nar\/gky354","article-title":"CRISPOR: Intuitive guide selection for CRISPR\/Cas9 genome editing experiments and screens","volume":"46","author":"Concordet","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"811","DOI":"10.1007\/s13238-017-0475-6","article-title":"Correction of beta-thalassemia mutant by base editor in human embryos","volume":"8","author":"Liang","year":"2017","journal-title":"Protein Cell"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1186\/s40348-018-0086-1","article-title":"Gene correction of HBB mutations in CD34(+) hematopoietic stem cells using Cas9 mRNA and ssODN donors","volume":"5","author":"Antony","year":"2018","journal-title":"Mol. Cell Pediatr."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"2060","DOI":"10.1073\/pnas.1620874114","article-title":"Barcoded nanoparticles for high throughput in vivo discovery of targeted therapeutics","volume":"114","author":"Dahlman","year":"2017","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"2148","DOI":"10.1021\/acs.nanolett.8b00432","article-title":"A Direct Comparison of in Vitro and in Vivo Nucleic Acid Delivery Mediated by Hundreds of Nanoparticles Reveals a Weak Correlation","volume":"18","author":"Paunovska","year":"2018","journal-title":"Nano Lett."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"8341","DOI":"10.1021\/acsnano.8b03640","article-title":"Analyzing 2000 in Vivo Drug Delivery Data Points Reveals Cholesterol Structure Impacts Nanoparticle Delivery","volume":"12","author":"Paunovska","year":"2018","journal-title":"ACS Nano"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"17095","DOI":"10.1021\/jacs.8b08976","article-title":"Nanoparticles That Deliver RNA to Bone Marrow Identified by in Vivo Directed Evolution","volume":"140","author":"Sago","year":"2018","journal-title":"J. Am. Chem. Soc."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"7197","DOI":"10.1039\/C8TB01642A","article-title":"Barcoding chemical modifications into nucleic acids improves drug stability in vivo","volume":"6","author":"Sago","year":"2018","journal-title":"J. Mater. Chem. B"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"E9944","DOI":"10.1073\/pnas.1811276115","article-title":"High-throughput in vivo screen of functional mRNA delivery identifies nanoparticles for endothelial cell gene editing","volume":"115","author":"Sago","year":"2018","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"e1902798","DOI":"10.1002\/adma.201902798","article-title":"Using Large Datasets to Understand Nanotechnology","volume":"31","author":"Paunovska","year":"2019","journal-title":"Adv. Mater."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1038\/s41551-020-00622-8","article-title":"A web tool for the design of prime-editing guide RNAs","volume":"5","author":"Chow","year":"2021","journal-title":"Nat. Biomed. Eng."},{"key":"ref_52","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\/23\/11\/5948\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:18:42Z","timestamp":1760138322000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/11\/5948"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,5,25]]},"references-count":52,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2022,6]]}},"alternative-id":["ijms23115948"],"URL":"https:\/\/doi.org\/10.3390\/ijms23115948","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,5,25]]}}}