Abstract
Pathogenic missense mutations in the alpha actin isotype 2 (ACTA2) gene cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection and death in childhood. Here we perform mutation-specific protein engineering to develop a bespoke CRISPR–Cas9 enzyme with enhanced on-target activity against the most common MSMDS-causative mutation ACTA2 R179H. To directly correct the R179H mutation, we screened dozens of configurations of base editors to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We create a murine model of MSMDS that shows phenotypes consistent with human patients, including vasculopathy and premature death, to explore the in vivo therapeutic potential of this strategy. Delivery of the customized base editor via an engineered smooth muscle-tropic adeno-associated virus (AAV-PR) vector substantially prolongs survival and rescues systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta and brain. Our results highlight how bespoke mutant-specific CRISPR–Cas9 enzymes can improve mutation correction with base editors.
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Data availability
Primary datasets are available in Supplementary Tables 1 and 5–7. Sequencing datasets are available with the NCBI Sequence Read Archive (SRA) under PRJNA1280096 (ref. 84). Plasmids from this study are available through Addgene.
References
Cw, T. et al. Heart disease and stroke statistics-2023 update: a report from the American Heart Association. Circulation 147, e93–e621 (2023).
Ostrem, B. E. L., Godfrey, D., Caruso, P. A. & Musolino, P. L. Monogenic causes of cerebrovascular disease in childhood: a case series. Pediatr. Neurol. 149, 39–43 (2023).
Jankovic, M. et al. The genetic basis of strokes in pediatric populations and insight into new therapeutic options. Int. J. Mol. Sci. 23, 1601 (2022).
Grossi, A. et al. Targeted re-sequencing in pediatric and perinatal stroke. Eur. J. Med. Genet. 63, 104030 (2020).
Ilinca, A. et al. A stroke gene panel for whole-exome sequencing. Eur. J. Hum. Genet. 27, 317–324 (2019).
Munot, P. et al. A novel distinctive cerebrovascular phenotype is associated with heterozygous Arg179 ACTA2 mutations. Brain J. Neurol. 135, 2506–2514 (2012).
Milewicz, D. M. et al. De novo ACTA2 mutation causes a novel syndrome of multisystemic smooth muscle dysfunction. Am. J. Med. Genet. A. 152A, 2437–2443 (2010).
Richer, J. et al. R179H mutation in ACTA2 expanding the phenotype to include prune-belly sequence and skin manifestations. Am. J. Med. Genet. A. 158A, 664–668 (2012).
Regalado, E. S. et al. Clinical history and management recommendations of the smooth muscle dysfunction syndrome due to ACTA2 arginine 179 alterations. Genet. Med. 20, 1206–1215 (2018).
Lauer, A. et al. Cerebrovascular disease progression in patients with ACTA2 Arg179 pathogenic variants. Neurology 96, e538–e552 (2021).
Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).
Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).
Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).
Gaudelli, N. M. et al. Directed evolution of adenine base editors with increased activity and therapeutic application. Nat. Biotechnol. 38, 892–900 (2020).
Richter, M. F. et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat. Biotechnol. 38, 883–891 (2020).
Huang, T. P., Newby, G. A. & Liu, D. R. Precision genome editing using cytosine and adenine base editors in mammalian cells. Nat. Protoc. 16, 1089–1128 (2021).
Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 368, 290–296 (2020).
Miller, S. M. et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat. Biotechnol. 38, 471–481 (2020).
Nishimasu, H. et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361, 1259–1262 (2018).
Kleinstiver, B. P. et al. Engineered CRISPR–Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).
Alves, C. R. R. et al. Optimization of base editors for the functional correction of SMN2 as a treatment for spinal muscular atrophy. Nat. Biomed. Eng. 8, 118–131 (2024).
Bzhilyanskaya, V. et al. High-fidelity PAMless base editing of hematopoietic stem cells to treat chronic granulomatous disease. Sci. Transl. Med. 16, eadj6779 (2024).
Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).
Porto, E. M., Komor, A. C., Slaymaker, I. M. & Yeo, G. W. Base editing: advances and therapeutic opportunities. Nat. Rev. Drug Discov. 19, 839–859 (2020).
Porto, E. M. & Komor, A. C. In the business of base editors: evolution from bench to bedside. PLoS Biol. 21, e3002071 (2023).
Christie, K. A. & Kleinstiver, B. P. Making the cut with PAMless CRISPR-Cas enzymes. Trends Genet. 37, 1053–1055 (2021).
Chatterjee, P., Jakimo, N. & Jacobson, J. M. Minimal PAM specificity of a highly similar SpCas9 ortholog. Sci. Adv. 4, eaau0766 (2018).
Zhao, L. et al. PAM-flexible genome editing with an engineered chimeric Cas9. Nat. Commun. 14, 6175 (2023).
Chai, A. C. et al. Base editing correction of hypertrophic cardiomyopathy in human cardiomyocytes and humanized mice. Nat. Med. 29, 401–411 (2023).
Lebek, S. et al. Ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing as a therapy for cardiac disease. Science 379, 179–185 (2023).
Hibshman, G. N. et al. Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9. Nat. Commun. 15, 3663 (2024).
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
Kleinstiver, B. P. et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).
Spencer, J. M. & Zhang, X. Deep mutational scanning of S. pyogenes Cas9 reveals important functional domains. Sci. Rep. 7, 16836 (2017).
Anders, C., Bargsten, K. & Jinek, M. Structural plasticity of PAM recognition by engineered variants of the rna-guided endonuclease Cas9. Mol. Cell 61, 895–902 (2016).
Walton, R. T., Hsu, J. Y., Joung, J. K. & Kleinstiver, B. P. Scalable characterization of the PAM requirements of CRISPR–Cas enzymes using HT-PAMDA. Nat. Protoc. 16, 1511–1547 (2021).
Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843–846 (2018).
Kim, H. S., Jeong, Y. K., Hur, J. K., Kim, J.-S. & Bae, S. Adenine base editors catalyze cytosine conversions in human cells. Nat. Biotechnol. 37, 1145–1148 (2019).
Jeong, Y. K. et al. Adenine base editor engineering reduces editing of bystander cytosines. Nat. Biotechnol. 39, 1426–1433 (2021).
Arbab, M. et al. Determinants of base editing outcomes from target library analysis and machine learning. Cell 182, 463–480.e30 (2020).
Kim, N. et al. Deep learning models to predict the editing efficiencies and outcomes of diverse base editors. Nat. Biotechnol. 42, 484–497 (2024).
Lino et al. An HDAC9-MALAT1-BRG1 complex mediates smooth muscle dysfunction in thoracic aortic aneurysm. Nat. Commun. 9, 1009 (2018).
Malhotra, R. et al. HDAC9 is implicated in atherosclerotic aortic calcification and affects vascular smooth muscle cell phenotype. Nat. Genet. 51, 1580–1587 (2019).
Lino Cardenas, C. L. et al. HDAC9 complex inhibition improves smooth muscle-dependent stenotic vascular disease. JCI Insight 4, e124706 (2019).
Chou, E. L. et al. Aortic cellular diversity and quantitative genome-wide association study trait prioritization through single-nuclear RNA sequencing of the aneurysmal human aorta. Arterioscler. Thromb. Vasc. Biol. 42, 1355–1374 (2022).
Bae, S., Park, J. & Kim, J.-S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).
Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).
Malinin, N. L. et al. Defining genome-wide CRISPR-Cas genome-editing nuclease activity with GUIDE-seq. Nat. Protoc. 16, 5592–5615 (2021).
Lazzarotto, C. R. et al. Population-scale cellular GUIDE-seq-2 and biochemical CHANGE-seq-R profiles reveal human genetic variation frequently affects Cas9 off-target activity. Preprint at bioRxiv https://doi.org/10.1101/2025.02.10.637517 (2025).
Lazzarotto, C. R. et al. CHANGE-seq-BE enables simultaneously sensitive and unbiased in vitro profiling of base editor genome-wide activity. Preprint at bioRxiv https://doi.org/10.1101/2024.03.28.586621 (2024).
Lazzarotto, C. R. et al. CHANGE-seq reveals genetic and epigenetic effects on CRISPR–Cas9 genome-wide activity. Nat. Biotechnol. 38, 1317–1327 (2020).
Xin, H.-B., Deng, K.-Y., Rishniw, M., Ji, G. & Kotlikoff, M. I. Smooth muscle expression of Cre recombinase and eGFP in transgenic mice. Physiol. Genomics 10, 211–215 (2002).
Liu, Z. et al. Vascular disease-causing mutation, smooth muscle α-actin R258C, dominantly suppresses functions of α-actin in human patient fibroblasts. Proc. Natl Acad. Sci. USA 114, E5569–E5578 (2017).
Koblan, L. W. et al. In vivo base editing rescues Hutchinson–Gilford progeria syndrome in mice. Nature 589, 608–614 (2021).
Levy, J. M. et al. Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. Nat. Biomed. Eng. 4, 97–110 (2020).
Ramirez, S. H. et al. An engineered adeno-associated virus capsid mediates efficient transduction of pericytes and smooth muscle cells of the brain vasculature. Hum. Gene Ther. 34, 682–696 (2023).
Kim, Y. et al. Gene therapy in cardiovascular disease: recent advances and future directions in science: a science advisory from the American Heart Association. Circulation 150, e471–e480 (2024).
Kleinstiver, B. & Walton, R. T. Crispr-Cas enzymes with enhanced on-target activity (2021).
Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).
Kleinstiver, B. P. et al. Broadening the targeting range of Staphylococcus aureus CRISPR–Cas9 by modifying PAM recognition. Nat. Biotechnol. 33, 1293–1298 (2015).
Kleinstiver, B. P. et al. Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. 37, 276–282 (2019).
Chen, Y. et al. Synergistic engineering of CRISPR–Cas nucleases enables robust mammalian genome editing. Innovation 3, 100264 (2022).
McGaw, C. et al. Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing. Nat. Commun. 13, 2833 (2022).
Zhang, H. et al. An engineered xCas12i with high activity, high specificity, and broad PAM range. Protein Cell 14, 540–545 (2023).
Yan, H. et al. Assessing and engineering the IscB-ωRNA system for programmed genome editing. Nat. Chem. Biol. 20, 1617–1628 (2024).
Wu, T. et al. An engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity. Nat. Chem. Biol. 19, 1384–1393 (2023).
Xiao, Q. et al. Engineered IscB-ωRNA system with expanded target range for base editing. Nat. Chem. Biol. 21, 100–108 (2025).
Silverstein, R. A. et al. Custom CRISPR-Cas9 PAM variants via scalable engineering and machine learning. Nature 643, 539–550 (2025).
Musunuru, K. et al. Patient-specific in vivo gene editing to treat a rare genetic disease. N. Engl. J. Med. 392, 2235–2243 (2025).
Lin, J. et al. Adenine base editing-mediated exon skipping restores dystrophin in humanized Duchenne mouse model. Nat. Commun. 15, 5927 (2024).
Davis, J. R. et al. Efficient in vivo base editing via single adeno-associated viruses with size-optimized genomes encoding compact adenine base editors. Nat. Biomed. Eng. 6, 1272–1283 (2022).
Zhang, H. et al. Adenine base editing in vivo with a single adeno-associated virus vector. GEN Biotechnol. 1, 285–299 (2022).
Liu, Z. et al. An all-in-one AAV vector for cardiac-specific gene silencing by an adenine base editor. 2024.09.30.615742 Preprint at bioRxiv https://doi.org/10.1101/2024.09.30.615742 (2024).
Tsuchida, C. A., Wasko, K. M., Hamilton, J. R. & Doudna, J. A. Targeted nonviral delivery of genome editors in vivo. Proc. Natl Acad. Sci. USA 121, e2307796121 (2024).
Kim, J., Eygeris, Y., Ryals, R. C., Jozic, A. & Sahay, G. Strategies for non-viral vectors targeting organs beyond the liver. Nat. Nanotechnol. 19, 428–447 (2024).
Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).
Rohland, N. & Reich, D. Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res. 22, 939–946 (2012).
Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).
DeRosa, S. et al. MCOLN1 gene therapy corrects neurologic dysfunction in the mouse model of mucolipidosis IV. Hum. Mol. Genet. 30, 908–922 (2021).
Lapinaite, A. et al. DNA capture by a CRISPR-Cas9-guided adenine base editor. Science 369, 566–571 (2020).
Kim, D., Kim, D., Lee, G., Cho, S.-I. & Kim, J.-S. Genome-wide target specificity of CRISPR RNA-guided adenine base editors. Nat. Biotechnol. 37, 430–435 (2019).
Hanlon, K. S. et al. Selection of an efficient AAV vector for robust CNS transgene expression. Mol. Ther. Methods Clin. Dev. 15, 320–332 (2019).
Alves, C. R. R. et al. Treatment of a severe vascular disease using a bespoke CRISPR-Cas9 base editor in mice. NCBI https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1280096 (2025).
Zettler, J., Schütz, V. & Mootz, H. D. The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Lett. 583, 909–914 (2009).
Acknowledgements
This work was supported by a Charles A. King Trust Postdoctoral Research Fellowship, Bank of America, N.A., Co-Trustees (C.R.R.A.), a James L. and Elisabeth C. Gamble Endowed Fund for Neuroscience Research/Mass General Neuroscience Transformative Scholar Award (C.R.R.A.), a MGH Physician/Scientist Development Award (C.R.R.A.), a Ministry of Science and ICT and National Research Foundation of Korea Award (RS-2024-00359396, to K.R.), a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship–Doctoral Postgraduate Scholarship–Doctoral (PGS D – 567791 to R.A.S.), an EMBO Long Term Fellowship (ALTF 750-2022, to J.F.d.S.), a Swiss National Science Foundation grant (P180777, to F.M.C.B.), St. Jude Children’s Research Hospital, American Lebanese Syrian Associated Charities (ALSAC), and National Institutes of Allergy and Infectious Diseases awards U01AI176470 and U01AI176471 (S.Q.T.), an MGH Howard M. Goodman Fellowship (to B.P.K.), the Kayden–Lambert MGH Research Scholar Award 2023-2028 (B.P.K.), a sponsored research agreement with Angea Biotherapeutics (R.M., D.Y.C., C.A.M., M.E.L., B.P.K. and P.L.M.) and National Institutes of Health grants K01NS134784 (C.R.R.A.), R01HL162928 (R.M.), K08NS112601 (D.Y.C.), R35GM142553 (L.H.C.), DC017117 (C.A.M.), DP2CA281401 (B.P.K.), P01HL142494 (B.P.K.) and R01NS125353 (to P.L.M., M.E.L. and B.P.K.). Some elements of Figs. 1a and 4c and Supplementary Fig. 35a were adapted from Servier Medical Art under a Creative Commons license CC BY 4.0.
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C.R.R.A., S.D. and V.K. contributed equally. L.L.H., L.R.F., H.E.S., C.E.S., P.K., S. McCarthy and C.L.L.C. contributed equally. M.E.L., B.P.K. and P.L.M. conceived of and designed the study. All authors designed, performed or supervised experiments and/or analysed data. C.R.R.A., C.L.L.C., L.L.H., H.E.S., P.K., L.R.F., S. McCarthy and S. Mitra performed cell culture experiments. C.R.R.A., S.D., V.K., C.L.L.C., L.L.H., H.E.S., C.E.S., P.K., L.R.F., C.E.F., S. McCarthy, S. Mitra and S.Y. performed molecular and biochemical experiments. S.D., V.K., C.L.L.C., C.E.S., C.E.F., T.I., J.L., R.R., K.R., R.M. and D.Y.C. conducted in vivo experiments in mice or performed histological or data analyses. C.R.R.A., L.L.H., H.E.S., L.R.F., S.Y. and J.F.d.S. performed plasmid cloning and lentivirus production. N.K. and R.A.S. performed HT-PAMDA experiments. D.d.l.C., J.X., H.L.G.-E. and C.A.M. performed titrations of AAV preparations and/or advised on AAV-related experiments. L.H.C. and F.M.C.B. performed protein purification. S.Q.T. and R.K.W. designed and performed CHANGE-seq-BE experiments. K.R., C.R.R.A., M.E.L. and B.P.K. wrote the paper with contributions or revisions from all authors.
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C.L.L.C., R.M., C.A.M., D.Y.C., B.P.K., M.E.L. and P.L.M. are inventors on a patent application filed by MGB that describes the development of genome-editing technologies to treat MSMDS. C.R.R.A., R.A.S., J.F.d.S. and B.P.K. are inventors on additional patents or patent applications filed by MGB that describe genome engineering technologies. C.R.R.A. is a consultant for Biogen and Ilios Therapeutics. S.Q.T. is an inventor on a patent covering CHANGE-seq. S.Q.T. is a member of the scientific advisory board of Prime Medicine and Ensoma. R.M., D.Y.C., C.A.M., B.P.K., M.E.L. and P.L.M. received sponsored research support from Angea Biotherapeutics, a company developing gene therapies for vasculopathies. R.M. receives research funding from Amgen, serves as a consultant for Pharmacosmos, Myokardia/BMS, Renovacor, Epizon Pharma and Third Pole, and performs speaker bureaus through Vox Media, all of which are unrelated to the current work. C.A.M. has financial interests in Chameleon Biosciences, Skylark Bio and Sphere Gene Therapeutics, companies developing adeno-associated virus vector technologies for gene therapy applications; C.A.M. performs paid consulting work for all three companies. C.A.M.’s interests were reviewed and are managed by MGH and MGB in accordance with their conflict-of-interest policies. B.P.K. is a consultant for EcoR1 capital, Novartis Venture Fund, Foresite Labs and Jumble Therapeutics, and is on the scientific advisory boards of Acrigen Biosciences, Life Edit Therapeutics and Prime Medicine. B.P.K. has a financial interest in Prime Medicine, a company developing therapeutic CRISPR–Cas technologies for gene editing. B.P.K.’s interests were reviewed and are managed by MGH and MGB in accordance with their conflict-of-interest policies. The other authors declare no competing interests.
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Supplementary information
Supplementary Information
Supplementary Notes 1 and 2, Figs. 1–35 and References.
Supplementary Table 1
Cas-OFFinder, GUIDE-seq2 and CHANGE-seq-BE results.
Supplementary Table 2
gRNA target sites.
Supplementary Table 3
Plasmids.
Supplementary Table 4
Oligonucleotides and probes.
Supplementary Table 5
rhAmpSeq results for gRNA A4.
Supplementary Table 6
rhAmpSeq results for gRNA A8.
Supplementary Table 7
Primary datasets.
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Alves, C.R.R., Das, S., Krishnan, V. et al. Treatment of a severe vascular disease using a bespoke CRISPR–Cas9 base editor in mice. Nat. Biomed. Eng (2025). https://doi.org/10.1038/s41551-025-01499-1
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DOI: https://doi.org/10.1038/s41551-025-01499-1
