HomeCirculationVol. 145, No. 10Efficient In Vivo Homology-Directed Repair Within Cardiomyocytes Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBEfficient In Vivo Homology-Directed Repair Within Cardiomyocytes Yanjiang Zheng, PhD, Nathan J. VanDusen, PhD, Catalina E. Butler, BS, Qing Ma, MD, Justin S. King, BS and William T. Pu, MD Yanjiang ZhengYanjiang Zheng Department of Biochemistry, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu, China (Y.Z.). , Nathan J. VanDusenNathan J. VanDusen Correspondence to: Nathan J. VanDusen, PhD, or William T. Pu, MD, 300 Longwood Ave, Boston, MA 02115. Email E-mail Address: [email protected] or E-mail Address: [email protected] https://orcid.org/0000-0002-2636-7228 Department of Cardiology, Boston Children’s Hospital, MA (Y.Z., N.J.V., C.E.B., Q.M., J.S.K., W.T.P.). , Catalina E. ButlerCatalina E. Butler https://orcid.org/0000-0002-0096-0197 Department of Cardiology, Boston Children’s Hospital, MA (Y.Z., N.J.V., C.E.B., Q.M., J.S.K., W.T.P.). , Qing MaQing Ma Department of Cardiology, Boston Children’s Hospital, MA (Y.Z., N.J.V., C.E.B., Q.M., J.S.K., W.T.P.). , Justin S. KingJustin S. King Department of Cardiology, Boston Children’s Hospital, MA (Y.Z., N.J.V., C.E.B., Q.M., J.S.K., W.T.P.). and William T. PuWilliam T. Pu https://orcid.org/0000-0002-4551-8079 Department of Cardiology, Boston Children’s Hospital, MA (Y.Z., N.J.V., C.E.B., Q.M., J.S.K., W.T.P.). Harvard Stem Cell Institute, Cambridge, MA (W.T.P.). Originally published7 Mar 2022https://doi.org/10.1161/CIRCULATIONAHA.120.052383Circulation. 2022;145:787–789CRISPR/Cas9-based genome editing technologies provide powerful tools for genetic manipulation. Cas9 in vivo genome editing of cardiomyocytes through nonhomologous end joining efficiently creates insertion–deletion mutations at guide RNA–targeted sites.1 However, precise addition of new genetic information, or mutation correction, requires alternate strategies. Delivery of Cas9 and a homology directed repair (HDR) template using adeno-associated virus (AAV) was recently shown to enable creation of precise genomic edits, even within postmitotic cells.2 We studied CRISPR/Cas9 and AAV-based homology directed repair (CASAAV-HDR) in cardiomyocytes. Animal experiments were approved by the Institutional Animal Care and Use Committee and adhered to institutional guidelines. The authors will provide data, results, and reagents on reasonable request.We studied CASAAV-HDR at Myl2 (MLC2v), a highly expressed, ventricle-specific sarcomere gene. We constructed AAV9 with an Myl2-specific guide RNA and a promoterless HDR template that replaces the native stop codon with self-cleaving 2A peptide followed by mScarlet, a red fluorescent protein (Figure [A]). Subcutaneous delivery of the AAV to postnatal day 0 mice with cardiac-restricted Cas9 expression (Tnnt2Cre;Rosa26fsCas9-P2A-GFP) yielded strong mScarlet expression in postnatal day 7 ventricular cardiomyocytes (Figure [Bi and C]). mScarlet expression required Cas9 and Myl2 homology arms (Figure [Bii and Biii]). AAV-delivered Cas9 successfully directed mScarlet expression, albeit with reduced efficiency compared with Rosa26fsCas9-P2A-GFP (Figure [Biv]). Guide RNAs targeting sequences on either side of the stop codon had equivalent performance (data not shown). We did not detect mScarlet in atrial cardiomyocytes, consistent with the targeted Myl2 allele retaining its expression pattern (Figure [C], top). Parallel experiments targeting atrial-specific Myl7 (MLC2a) resulted in mScarlet expression within atrial but not ventricular cardiomyocytes (Figure [C], bottom). Flow cytometry quantification of Myl2 knockin efficiency after systemic AAV injection showed that ≈45% of ventricular cardiomyocytes displayed strong fluorescence, with steep dose response (Figure [D]). In atrial cardiomyocytes, Myl7 knockin efficiency at the cellular level was 20% and had similar dose response (Figure [D]).Download figureDownload PowerPointFigure. CASAAV-HDR targeted transgene insertion is highly efficient in neonatal and terminally differentiated cardiomyocytes in vivo.Unless noted otherwise, adeno-associated virus (AAV) was delivered subcutaneously at postnatal day 0 (P0) and hearts were analyzed at postnatal day 7 (P7). A, CRISPR/Cas9/AAV9-based somatic mutagenesis (CASAAV)–homology directed repair (HDR) strategy to insert P2A-mScarlet at the stop codon of endogenous Myl2. Cas9 was expressed specifically in cardiomyocytes in Tnnt2Cre;Rosa26fsCas9-P2A-GFP mice. Cas9-induced double strand breaks can be repaired by HDR or nonhomologous end joining (NHEJ). Blue lines, homology arms; open arrows, primers used for amplicon sequencing. Unless noted otherwise, AAV9 was delivered subcutaneously at P0 and hearts were analyzed at P7. B, CASAAV-HDR integration of P2A-mScarlet at Myl2. i, AAV9-HDR-Myl2, CASAAV-HDR vector targeting Myl2, was delivered to Tnnt2Cre;Rosa26fsCas9-P2A-GFP mice. ii, No Cas9 vector was delivered without Cas9 expression. iii, AAV similar to AAV9-HDR-Myl2 but lacking homology arms was delivered to Tnnt2Cre;Rosa26fsCas9-P2A-GFP mice. iv, AAV-Cas9, AAV9-HDR-Myl2, plus AAV9-Tnnt2-Cas9 was delivered to wild-type mice. Scale bar, 50 µm. C, CASAAV-HDR vectors targeting Myl2 or Myl7 resulted in mScarlet expression specifically in ventricular or atrial chambers. Scale bar, 50 µm. D, CASAAV-HDR dose response. AAV was administered at high, middle, and low doses (5×1011, 5×1010, and 5×109 vg/g, respectively), resulting in ≈96%, 83%, and 51% myocardial transduction, respectively. Cardiomyocytes, dissociated by Langendorff perfusion, were analyzed for GFP expression by flow cytometry. E, Quantification of mutations induced by high-dose CASAAV-HDR insertion of P2A-mScarlet into the C terminus of Myl2 or Myl7. The junctions between inserted sequence and endogenous sequence were amplified from cDNA. Primers are illustrated in A. For alleles lacking an insert, a fragment was amplified from DNA using primers flanking the guide RNA target site. Amplicons were deeply sequenced and analyzed for the indicated types of modifications. F, Myl2 HDR efficiency in fetal, neonatal, or mature cardiomyocytes. AAV was administered at equivalent middle dose (5×1010 vg/g; E15.5 embryo = 0.6 g) at each stage, resulting in 80% to 83% myocardial transduction. mScarlet-expressing cardiomyocytes were quantified by flow cytometry. Differences between groups were not significant. G, Summary of HDR efficiency at 9 different loci, as a function of gene expression level in P0 ventricular cardiomyocytes, or atrial cardiomyocytes in the case of Myl7. Homology arms were ≈1 kb long. Shading indicates 95% CI for fitted line. H, Adult heart with insertion of mScarlet into the Pln locus after P0 administration of CASAAV-HDR vector. Scale bar, 200 µm. I, In situ imaging showing localization of mScarlet fused to Pln or Ttn in mature cardiomyocytes. Scale bar, 10 µm. **Dunnett P<0.001. *P<0.01. Error bars reflect standard error.We quantified mutations created during the CASAAV-HDR DNA repair process (Figure [E]). The 5′ and 3′ junctions between the inserted template and the endogenous Myl2 or Myl7 sequences were amplified from RNA and deeply sequenced. We also quantified mutations in alleles that did not contain an inserted template by amplifying and sequencing cardiomyocyte genomic DNA flanking the guide RNA target site. For Myl2, 95.9% and 99.4% of transcripts containing the inserted template had the expected 5′ or 3′ junction sequences, respectively, whereas 11.3% of alleles lacking an insert contained a mutation, reflecting nonhomologous end joining (Figure [E]). For Myl7, these numbers were 85.8%, 97.8%, and 27.1%, respectively (Figure [E]). These data indicate that CASAAV-HDR insertion is precise and that a subset of alleles without repair template integration contained nonhomologous end joining–induced mutations. Integration of AAV-inverted terminal repeats at Myl2 or Myl7 was detected in <2% of sequences (Figure [E]). Inverted terminal repeat sequencing found inverted terminal repeat integration elsewhere in the genome 4- to 28-fold less frequently than at Myl2.Although HDR has been thought to be limited to proliferating cells,3 CASAAV-HDR occurred in postmitotic neurons and in postmitotic adult cardiomyocytes.2,4 We assessed the effect of cardiomyocyte proliferation on CASAAV-HDR efficiency by measuring CASAAV-HDR at Myl2 at different developmental stages. CASAAV-HDR efficiency at the cellular level was comparable when AAV was delivered to fetal, neonatal, or mature mice (Figure [F]). Because of technical limitations, we were unable to quantify the fraction of cardiomyocyte genomes successfully modified by HDR and make comparisons among AAV delivery times at the genome level.We targeted 7 additional loci—Yap1, Tmem43, Nfatc3, Bdh1, Mkl1, Ttn, and Pln—fusing either an HA tag or mScarlet to each. Insertion efficiency varied dramatically between loci, with HDR efficiency at the cellular level generally correlating with target gene expression (Figure [G]). The 5 lowly expressed genes (<5 transcripts per million) had low HDR efficiency, whereas ≥20% of cardiomyocytes were edited in each of the 4 robustly expressed genes (>100 transcripts per million; Figure [G and H]). TTN-mScarlet and mScarlet-PLN fusion proteins localized to the sarcomere and sarcoplasmic reticulum, respectively, consistent with the localization of the endogenous proteins (Figure [I]).Systemic delivery of CASAAV-HDR vectors achieved efficient and precise in vivo somatic genome modification that did not require cardiomyocyte proliferation. Efficiency correlated with expression level of the target gene and in the best case reached remarkably high levels (45% of cardiomyocytes). While this article was in preparation, limited success with CASAAV-HDR in the heart was reported, although HDR efficiency was low and required direct intramyocardial injection because systemic delivery was unsuccessful.5 We successfully used CASAAV-HDR to monitor protein localization and anticipate it will be useful for many other applications, such as precise introduction of mutations to model disease or probe gene function. CASAAV-HDR may also enable efficient, permanent, and precisely targeted delivery of therapeutic transgenes to validated loci. We envision future studies will further expand research and translational applications by identifying attributes of loci that make them amenable to efficient HDR.Article InformationSources of FundingDr Zheng was funded by the Chinese Scholarship Council. Drs VanDusen (grant K99HL143194) and Pu (grants 2UM1 HL098166 and R01 HL146634) were supported by the National Institutes of Health/National Heart, Lung, and Blood Institute.Nonstandard Abbreviations and AcronymsAAVadeno-associated virusCASAAVCRISPR/Cas9/AAV9-based somatic mutagenesisHDRhomology directed repairDisclosures None.Footnotes*Y. Zheng and N.J. VanDusen contributed equally.For Sources of Funding and Disclosures, see page 789.Circulation is available at www.ahajournals.org/journal/circCorrespondence to: Nathan J. VanDusen, PhD, or William T. Pu, MD, 300 Longwood Ave, Boston, MA 02115. Email nathan.[email protected]harvard.edu or william.[email protected]chboston.orgReferences1. Guo Y, VanDusen NJ, Zhang L, Gu W, Sethi I, Guatimosim S, Ma Q, Jardin BD, Ai Y, Zhang D, et al.. Analysis of cardiac myocyte maturation using CASAAV, a platform for rapid dissection of cardiac myocyte gene function in vivo.Circ Res. 2017; 120:1874–1888. doi: 10.1161/CIRCRESAHA.116.310283LinkGoogle Scholar2. Nishiyama J, Mikuni T, Yasuda R. Virus-mediated genome editing via homology-directed repair in mitotic and postmitotic cells in mammalian brain.Neuron. 2017; 96:755–768.e5. doi: 10.1016/j.neuron.2017.10.004CrossrefMedlineGoogle Scholar3. Yeh CD, Richardson CD, Corn JE. Advances in genome editing through control of DNA repair pathways.Nat Cell Biol. 2019; 21:1468–1478. doi: 10.1038/s41556-019-0425-zCrossrefMedlineGoogle Scholar4. Ishizu T, Higo S, Masumura Y, Kohama Y, Shiba M, Higo T, Shibamoto M, Nakagawa A, Morimoto S, Takashima S, et al.. Targeted genome replacement via homology-directed repair in non-dividing cardiomyocytes.Sci Rep. 2017; 7:9363. doi: 10.1038/s41598-017-09716-xCrossrefMedlineGoogle Scholar5. Kohama Y, Higo S, Masumura Y, Shiba M, Kondo T, Ishizu T, Higo T, Nakamura S, Kameda S, Tabata T, et al.. Adeno-associated virus-mediated gene delivery promotes S-phase entry-independent precise targeted integration in cardiomyocytes.Sci Rep. 2020; 10:15348. doi: 10.1038/s41598-020-72216-yCrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited ByLiu N and Olson E (2022) CRISPR Modeling and Correction of Cardiovascular Disease, Circulation Research, 130:12, (1827-1850), Online publication date: 10-Jun-2022. March 8, 2022Vol 145, Issue 10Article InformationMetrics © 2022 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.120.052383PMID: 35254919 Originally publishedMarch 7, 2022 Keywordsmyocytes, cardiacgenome editingCRISPR-associated protein 9homology-directed dsDNA break repairPDF download Advertisement SubjectsAnimal Models of Human DiseaseGene TherapyGenetically Altered and Transgenic Models