Skip main navigation

CRISPR Activation Reverses Haploinsufficiency and Functional Deficits Caused by TTN Truncation Variants

Originally published 2024;149:1285–1297


    TTN truncation variants (TTNtvs) are the most common genetic lesion identified in individuals with dilated cardiomyopathy, a disease with high morbidity and mortality rates. TTNtvs reduce normal TTN (titin) protein levels, produce truncated proteins, and impair sarcomere content and function. Therapeutics targeting TTNtvs have been elusive because of the immense size of TTN, the rarity of specific TTNtvs, and incomplete knowledge of TTNtv pathogenicity.


    We adapted CRISPR activation using dCas9-VPR to functionally interrogate TTNtv pathogenicity and develop a therapeutic in human cardiomyocytes and 3-dimensional cardiac microtissues engineered from induced pluripotent stem cell models harboring a dilated cardiomyopathy–associated TTNtv. We performed guide RNA screening with custom TTN reporter assays, agarose gel electrophoresis to quantify TTN protein levels and isoforms, and RNA sequencing to identify molecular consequences of TTN activation. Cardiomyocyte epigenetic assays were also used to nominate DNA regulatory elements to enable cardiomyocyte-specific TTN activation.


    CRISPR activation of TTN using single guide RNAs targeting either the TTN promoter or regulatory elements in spatial proximity to the TTN promoter through 3-dimensional chromatin interactions rescued TTN protein deficits disturbed by TTNtvs. Increasing TTN protein levels normalized sarcomere content and contractile function despite increasing truncated TTN protein. In addition to TTN transcripts, CRISPR activation also increased levels of myofibril assembly-related and sarcomere-related transcripts.


    TTN CRISPR activation rescued TTNtv-related functional deficits despite increasing truncated TTN levels, which provides evidence to support haploinsufficiency as a relevant genetic mechanism underlying heterozygous TTNtvs. CRISPR activation could be developed as a therapeutic to treat a large proportion of TTNtvs.


    Supplemental Material is available at

    *S. Ghahremani and A. Kanwal contributed equally.

    For Sources of Funding and Disclosures, see page 1295.

    Circulation is available at

    Correspondence to: J. Travis Hinson, MD, UConn Health and The Jackson Laboratory for Genomic Medicine, 10 Discovery Dr, Farmington, CT 06032. Email


    • 1. Hershberger RE, Hedges DJ, Morales A. Dilated cardiomyopathy: the complexity of a diverse genetic architecture.Nat Rev Cardiol. 2013; 10:531–547. doi: 10.1038/nrcardio.2013.105CrossrefMedlineGoogle Scholar
    • 2. Hershberger RE, Jordan E. Dilated cardiomyopathy overview. In: Adam MP, Everman DB, Mirzaa GM, et al, eds. GeneReviews. University of Washington: 1993.Google Scholar
    • 3. Japp AG, Gulati A, Cook SA, Cowie MR, Prasad SK. The diagnosis and evaluation of dilated cardiomyopathy.J Am Coll Cardiol. 2016; 67:2996–3010. doi: 10.1016/j.jacc.2016.03.590CrossrefMedlineGoogle Scholar
    • 4. Roberts AM, Ware JS, Herman DS, Schafer S, Baksi J, Bick AG, Buchan RJ, Walsh R, John S, Wilkinson S, et al. Integrated allelic, transcriptional, and phenomic dissection of the cardiac effects of titin truncations in health and disease.Sci Transl Med. 2015; 7:270ra6. doi: 10.1126/scitranslmed.3010134CrossrefMedlineGoogle Scholar
    • 5. Herman DS, Lam L, Taylor MR, Wang L, Teekakirikul P, Christodoulou D, Conner L, DePalma SR, McDonough B, Sparks E, et al. Truncations of titin causing dilated cardiomyopathy.N Engl J Med. 2012; 366:619–628. doi: 10.1056/NEJMoa1110186CrossrefMedlineGoogle Scholar
    • 6. Goli R, Li J, Brandimarto J, Levine LD, Riis V, McAfee Q, DePalma S, Haghighi A, Seidman JG, Seidman CE, et al; IMAC-2 and IPAC Investigators. Genetic and phenotypic landscape of peripartum cardiomyopathy.Circulation. 2021; 143:1852–1862. doi: 10.1161/CIRCULATIONAHA.120.052395LinkGoogle Scholar
    • 7. Lota AS, Hazebroek MR, Theotokis P, Wassall R, Salmi S, Halliday BP, Tayal U, Verdonschot J, Meena D, Owen R, et al. Genetic architecture of acute myocarditis and the overlap with inherited cardiomyopathy.Circulation. 2022; 146:1123–1134. doi: 10.1161/CIRCULATIONAHA.121.058457LinkGoogle Scholar
    • 8. Hinson JT, Chopra A, Nafissi N, Polacheck WJ, Benson CC, Swist S, Gorham J, Yang L, Schafer S, Sheng CC, et al. Heart disease Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy.Science. 2015; 349:982–986. doi: 10.1126/science.aaa5458CrossrefMedlineGoogle Scholar
    • 9. Fomin A, Gartner A, Cyganek L, Tiburcy M, Tuleta I, Wellers L, Folsche L, Hobbach AJ, von Frieling-Salewsky M, Unger A, et al. Truncated titin proteins and titin haploinsufficiency are targets for functional recovery in human cardiomyopathy due to TTN mutations.Sci Transl Med. 2021; 13:eabd3079. doi: 10.1126/scitranslmed.abd3079CrossrefMedlineGoogle Scholar
    • 10. Schafer S, de Marvao A, Adami E, Fiedler LR, Ng B, Khin E, Rackham OJ, van Heesch S, Pua CJ, Kui M, et al. Titin-truncating variants affect heart function in disease cohorts and the general population.Nat Genet. 2017; 49:46–53. doi: 10.1038/ng.3719CrossrefMedlineGoogle Scholar
    • 11. Romano R, Ghahremani S, Zimmerman T, Legere N, Thakar K, Ladha FA, Pettinato AM, Hinson JT. Reading frame repair of TTN truncation variants restores titin quantity and functions.Circulation. 2022; 145:194–205. doi: 10.1161/CIRCULATIONAHA.120.049997LinkGoogle Scholar
    • 12. Hinson JT. Molecular genetic mechanisms of dilated cardiomyopathy.Curr Opin Genet Dev. 2022; 76:101959. doi: 10.1016/j.gde.2022.101959CrossrefMedlineGoogle Scholar
    • 13. Chopra A, Kutys ML, Zhang K, Polacheck WJ, Sheng CC, Luu RJ, Eyckmans J, Hinson JT, Seidman JG, Seidman CE, et al. Force generation via beta-cardiac myosin, titin, and alpha-actinin drives cardiac sarcomere assembly from cell-matrix adhesions.Dev Cell. 2018; 44:87–96.e5. doi: 10.1016/j.devcel.2017.12.012CrossrefMedlineGoogle Scholar
    • 14. McAfee Q, Chen CY, Yang Y, Caporizzo MA, Morley M, Babu A, Jeong S, Brandimarto J, Bedi KC, Flam E, et al. Truncated titin proteins in dilated cardiomyopathy.Sci Transl Med. 2021; 13:eabd7287. doi: 10.1126/scitranslmed.abd7287CrossrefMedlineGoogle Scholar
    • 15. Zhou H, Liu J, Zhou C, Gao N, Rao Z, Li H, Hu X, Li C, Yao X, Shen X, et al. In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR-dCas9-activator transgenic mice.Nat Neurosci. 2018; 21:440–446. doi: 10.1038/s41593-017-0060-6CrossrefMedlineGoogle Scholar
    • 16. Gemberling MP, Siklenka K, Rodriguez E, Tonn-Eisinger KR, Barrera A, Liu F, Kantor A, Li L, Cigliola V, Hazlett MF, et al. Transgenic mice for in vivo epigenome editing with CRISPR-based systems.Nat Methods. 2021; 18:965–974. doi: 10.1038/s41592-021-01207-2CrossrefMedlineGoogle Scholar
    • 17. Warren CM, Krzesinski PR, Greaser ML. Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins.Electrophoresis. 2003; 24:1695–1702. doi: 10.1002/elps.200305392CrossrefMedlineGoogle Scholar
    • 18. Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, Eswar PRI, Lin S, Kiani S, Guzman CD, Wiegand DJ, et al. Highly efficient Cas9-mediated transcriptional programming.Nat Methods. 2015; 12:326–328. doi: 10.1038/nmeth.3312CrossrefMedlineGoogle Scholar
    • 19. Schoger E, Carroll KJ, Iyer LM, McAnally JR, Tan W, Liu N, Noack C, Shomroni O, Salinas G, Gross J, et al. CRISPR-mediated activation of endogenous gene expression in the postnatal heart.Circ Res. 2020; 126:6–24. doi: 10.1161/CIRCRESAHA.118.314522LinkGoogle Scholar
    • 20. Farza H, Townsend PJ, Carrier L, Barton PJ, Mesnard L, Bahrend E, Forissier JF, Fiszman M, Yacoub MH, Schwartz K. Genomic organisation, alternative splicing and polymorphisms of the human cardiac troponin T gene.J Mol Cell Cardiol. 1998; 30:1247–1253. doi: 10.1006/jmcc.1998.0698CrossrefMedlineGoogle Scholar
    • 21. Donnelly MLL, Luke G, Mehrotra A, Li X, Hughes LE, Gani D, Ryan MD. Analysis of the aphthovirus 2A/2B polyprotein “cleavage” mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip.’J Gen Virol. 2001; 82:1013–1025. doi: 10.1099/0022-1317-82-5-1013CrossrefMedlineGoogle Scholar
    • 22. Pettinato AM, Yoo D, VanOudenhove J, Chen YS, Cohn R, Ladha FA, Yang X, Thakar K, Romano R, Legere N, et al. Sarcomere function activates a p53-dependent DNA damage response that promotes polyploidization and limits in vivo cell engraftment.Cell Rep. 2021; 35:109088. doi: 10.1016/j.celrep.2021.109088CrossrefMedlineGoogle Scholar
    • 23. Lian X, Zhang J, Azarin SM, Zhu K, Hazeltine LB, Bao X, Hsiao C, Kamp TJ, Palecek SP. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions.Nat Protoc. 2013; 8:162–175. doi: 10.1038/nprot.2012.150CrossrefMedlineGoogle Scholar
    • 24. Tohyama S, Hattori F, Sano M, Hishiki T, Nagahata Y, Matsuura T, Hashimoto H, Suzuki T, Yamashita H, Satoh Y, et al. Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes.Cell Stem Cell. 2013; 12:127–137. doi: 10.1016/j.stem.2012.09.013CrossrefMedlineGoogle Scholar
    • 25. Zou J, Tran D, Baalbaki M, Tang LF, Poon A, Pelonero A, Titus EW, Yuan C, Shi C, Patchava S, et al. An internal promoter underlies the difference in disease severity between N- and C-terminal truncation mutations of Titin in zebrafish.Elife. 2015; 4:e09406. doi: 10.7554/eLife.09406CrossrefMedlineGoogle Scholar
    • 26. Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond.Nat Rev Mol Cell Biol. 2012; 13:89–102. doi: 10.1038/nrm3270CrossrefMedlineGoogle Scholar
    • 27. Glembotski CC. Endoplasmic reticulum stress in the heart.Circ Res. 2007; 101:975–984. doi: 10.1161/CIRCRESAHA.107.161273LinkGoogle Scholar
    • 28. Feyen DAM, Perea-Gil I, Maas RGC, Harakalova M, Gavidia AA, Arthur Ataam J, Wu TH, Vink A, Pei J, Vadgama N, et al. Unfolded protein response as a compensatory mechanism and potential therapeutic target in PLN R14del cardiomyopathy.Circulation. 2021; 144:382–392. doi: 10.1161/CIRCULATIONAHA.120.049844LinkGoogle Scholar
    • 29. Wang S, Binder P, Fang Q, Wang Z, Xiao W, Liu W, Wang X. Endoplasmic reticulum stress in the heart: insights into mechanisms and drug targets.Br J Pharmacol. 2018; 175:1293–1304. doi: 10.1111/bph.13888CrossrefMedlineGoogle Scholar
    • 30. Oslowski CM, Urano F. Measuring ER stress and the unfolded protein response using mammalian tissue culture system.Methods Enzymol. 2011; 490:71–92. doi: 10.1016/B978-0-12-385114-7.00004-0CrossrefMedlineGoogle Scholar
    • 31. Borgia MB, Borgia A, Best RB, Steward A, Nettels D, Wunderlich B, Schuler B, Clarke J. Single-molecule fluorescence reveals sequence-specific misfolding in multidomain proteins.Nature. 2011; 474:662–665. doi: 10.1038/nature10099CrossrefMedlineGoogle Scholar
    • 32. Barkal AA, Srinivasan S, Hashimoto T, Gifford DK, Sherwood RI. Cas9 functionally opens chromatin.PLoS One. 2016; 11:e0152683. doi: 10.1371/journal.pone.0152683CrossrefMedlineGoogle Scholar
    • 33. Fullwood MJ, Wei CL, Liu ET, Ruan Y. Next-generation DNA sequencing of paired-end tags (PET) for transcriptome and genome analyses.Genome Res. 2009; 19:521–532. doi: 10.1101/gr.074906.107CrossrefMedlineGoogle Scholar
    • 34. Buenrostro JD, Wu B, Chang HY, Greenleaf WJ. ATAC-seq: a method for assaying chromatin accessibility genome-wide.Curr Protoc Mol Biol. 2015; 109:21 29 21–21 29 29. doi: 10.1002/0471142727.mb2129s109CrossrefMedlineGoogle Scholar
    • 35. Creyghton MP, Cheng AW, Welstead GG, Kooistra T, Carey BW, Steine EJ, Hanna J, Lodato MA, Frampton GM, Sharp PA, et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state.Proc Natl Acad Sci U S A. 2010; 107:21931–21936. doi: 10.1073/pnas.1016071107CrossrefMedlineGoogle Scholar
    • 36. Li G, Fullwood MJ, Xu H, Mulawadi FH, Velkov S, Vega V, Ariyaratne PN, Mohamed YB, Ooi HS, Tennakoon C, et al. ChIA-PET tool for comprehensive chromatin interaction analysis with paired-end tag sequencing.Genome Biol. 2010; 11:R22. doi: 10.1186/gb-2010-11-2-r22CrossrefMedlineGoogle Scholar
    • 37. Clippinger SR, Cloonan PE, Greenberg L, Ernst M, Stump WT, Greenberg MJ. Disrupted mechanobiology links the molecular and cellular phenotypes in familial dilated cardiomyopathy.Proc Natl Acad Sci U S A. 2019; 116:17831–17840. doi: 10.1073/pnas.1910962116CrossrefMedlineGoogle Scholar
    • 38. Ribeiro AJ, Ang YS, Fu JD, Rivas RN, Mohamed TM, Higgs GC, Srivastava D, Pruitt BL. Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness.Proc Natl Acad Sci U S A. 2015; 112:12705–12710. doi: 10.1073/pnas.1508073112CrossrefMedlineGoogle Scholar
    • 39. Cohn R, Thakar K, Lowe A, Ladha FA, Pettinato AM, Romano R, Meredith E, Chen YS, Atamanuk K, Huey BD, et al. A contraction stress model of hypertrophic cardiomyopathy due to sarcomere mutations.Stem Cell Rep. 2019; 12:71–83. doi: 10.1016/j.stemcr.2018.11.015CrossrefMedlineGoogle Scholar
    • 40. Loescher CM, Hobbach AJ, Linke WA. Titin (TTN): from molecule to modifications, mechanics, and medical significance.Cardiovasc Res. 2022; 118:2903–2918. doi: 10.1093/cvr/cvab328CrossrefMedlineGoogle Scholar
    • 41. Gramlich M, Pane LS, Zhou Q, Chen Z, Murgia M, Schotterl S, Goedel A, Metzger K, Brade T, Parrotta E, et al. Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy.EMBO Mol Med. 2015; 7:562–576. doi: 10.15252/emmm.201505047CrossrefMedlineGoogle Scholar
    • 42. Matharu N, Rattanasopha S, Tamura S, Maliskova L, Wang Y, Bernard A, Hardin A, Eckalbar WL, Vaisse C, Ahituv N. CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency.Science. 2019; 363:eaau0629. doi: 10.1126/science.aau0629CrossrefMedlineGoogle Scholar
    • 43. Gramlich M, Michely B, Krohne C, Heuser A, Erdmann B, Klaassen S, Hudson B, Magarin M, Kirchner F, Todiras M, et al. Stress-induced dilated cardiomyopathy in a knock-in mouse model mimicking human titin-based disease.J Mol Cell Cardiol. 2009; 47:352–358. doi: 10.1016/j.yjmcc.2009.04.014CrossrefMedlineGoogle Scholar
    • 44. Chuderland D, Seger R. Protein-protein interactions in the regulation of the extracellular signal-regulated kinase.Mol Biotechnol. 2005; 29:57–74. doi: 10.1385/MB:29:1:57CrossrefMedlineGoogle Scholar
    • 45. Gautel M, Castiglione Morelli MA, Pfuhl M, Motta A, Pastore A. A calmodulin-binding sequence in the C-terminus of human cardiac titin kinase.Eur J Biochem. 1995; 230:752–759.MedlineGoogle Scholar
    • 46. Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, et al. Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576:149–157. doi: 10.1038/s41586-019-1711-4CrossrefMedlineGoogle Scholar
    • 47. Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage.Nature. 2017; 551:464–471. doi: 10.1038/nature24644CrossrefMedlineGoogle Scholar
    • 48. Karbassi E, Fenix A, Marchiano S, Muraoka N, Nakamura K, Yang X, Murry CE. Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine.Nat Rev Cardiol. 2020; 17:341–359. doi: 10.1038/s41569-019-0331-xCrossrefMedlineGoogle Scholar
    • 49. Hinson JT, Chopra A, Lowe A, Sheng CC, Gupta RM, Kuppusamy R, O’Sullivan J, Rowe G, Wakimoto H, Gorham J, et al. Integrative analysis of PRKAG2 cardiomyopathy iPS and microtissue models identifies AMPK as a regulator of metabolism, survival, and fibrosis.Cell Rep. 2016; 17:3292–3304. doi: 10.1016/j.celrep.2016.11.066CrossrefMedlineGoogle Scholar
    • 50. Ng R, Manring H, Papoutsidakis N, Albertelli T, Tsai N, See CJ, Li X, Park J, Stevens TL, Bobbili PJ, et al. Patient mutations linked to arrhythmogenic cardiomyopathy enhance calpain-mediated desmoplakin degradation.JCI Insight. 2019; 5:e128643. doi: 10.1172/jci.insight.128643CrossrefMedlineGoogle Scholar


    eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.

    Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.