CRISPR/Cas9 mediated knockout of the DES gene alleles with desminopathy-related heterozygous gain-of-function mutations

Hereditary cardiomyopathies are characterized by the generally poor prognosis and low 5-year survival of patients with severe symptoms. Besides surgical approaches, cardiomyopathy therapy mainly palliative and often heart transplantation is the only option to improve patient state and prognosis. Some of these pathologies are associated with the autosomal-dominant DES gene mutations. DES encodes intermediate filaments protein desmin, which defects causes desminopathies involving most active muscles such as skeletal muscles, myocardium and respiratory muscles. New therapeutic based on genome editing approaches could be used to correct causative genetic defect. There are data that heterozygous nonsense mutations in DES gene may be asymptomatic. Thus there is, apparently, a possibility to decrease severity of desminopathy using mutant allele knockout. Purpose. The aim of this work was to test the possibility of specific knockout of the DES gene alleles with heterozygous desminopathy-associated mutations by means of genome editing methods. Materials. We received genetic materials of three patients with desminopathy caused by DES gene mutations (c.330_338del, p.A337P (c.1009G>C) и p.R355P (c.1064G>C)). Guide RNA, compatible with nucleases SaCas9 and eSpCas9(1.1) were designed using online service Benchling and cloned into plasmids with corresponding Cas9 nucleases. Editing plasmids were cotransfected into HEK293T cells with “target” plasmids, containing DES gene sites with mutations. NHEJ-produced indels were assessed using TIDE-analysis with amplified and sequenced sites or using T7E1 analysis. Results. Combination sgRNA for c.330_338del with eSpCas9(1.1) demonstrated most mean efficiency of 2,22% (up to 8,06%). Others combinations of sgRNAs and Cas9 efficiency did not overcome 3%. Conclusions. Achieved knockout efficiency is evidently not enough for organism-level desminopathy correction. The need for specific knockout of mutated alleles does not allow usage of different guide RNAs for CRISPR/Cas9, so it is necessary to improve the developed systems to increase their efficiency or to use new, more efficient, targeted nucleases.

cardiomyopathy, desminopathy, DES, desmin, genome editing, CRISPR/Cas9

1. McKenna W.J., Maron B.J., Thiene G. Classification, epidemiology, and global burden of cardiomyopathies. Circulation Research 2017;121:722-30. https://doi.org/10.1161/CIRCRESAHA.117.309711.

2. Elliott P., Andersson B., Arbustini E., et al. Classification of the cardiomyopathies: A position statement from the european society of cardiology working group on myocardial and pericardial diseases. European Heart Journal 2008;29:270-6. https://doi.org/10.1093/eurheartj/ehm342.

3. Westphal J.G., Rigopoulos A.G., Bakogiannis C., et al. The MOGE(S) classification for cardiomyopathies: current status and future outlook. Heart Failure Reviews 2017;22:743-52. https://doi.org/10.1007/s10741-017-9641-4.

4. Paulin D., Li Z. Desmin: A major intermediate filament protein essential for the structural integrity and function of muscle. Experimental Cell Research 2004;301:1-7. https://doi.org/10.1016/j.yexcr.2004.08.004.

5. Goldfarb L.G., Dalakas M.C. Tragedy in a heartbeat: Malfunctioning desmin causes skeletal and cardiac muscle disease. Journal of Clinical Investigation 2009;119:1806-13. https://doi.org/10.1172/JCI38027.

6. McLendon P.M., Robbins J. Desmin-related cardiomyopathy: An unfolding story. American Journal of Physiology - Heart and Circulatory Physiology 2011;301. https://doi.org/10.1152/ajpheart.00601.2011.

7. van Spaendonck-Zwarts K.Y., van Hessem L., Jongbloed J.D.H., et al. Desmin-related myopathy. Clinical Genetics 2011;80:354-66. https://doi.org/10.1111/j.1399-0004.2010.01512.x.

8. Li Z., Mericskay M., Agbulut O., et al. Desmin is essential for the tensile strength and integrity of myofibrils but not for myogenic commitment, differentiation, and fusion of skeletal muscle. Journal of Cell Biology 1997;139:129-44. https://doi.org/10.1083/jcb.139.1.129.

9. Goldfarb L.G., Park K.Y., Cervenákova L., et al. Missense mutations in desmin associated with familial cardiac and skeletal myopathy. Nature Genetics 1998;19:402-3. https://doi.org/10.1038/1300.

10. Arbustini E., Pasotti M., Pilotto A., et al. Desmin accumulation restrictive cardiomyopathy and atrioventricular block associated with desmin gene defects. European Journal of Heart Failure 2006;8:477-83. https://doi.org/10.1016/j.ejheart.2005.11.003.

11. HGMD® DES gene result n.d. http://www.hgmd.cf.ac.uk/ac/gene.php?gene=DES (accessed September 14, 2020).

12. Capetanaki Y., Papathanasiou S., Diokmetzidou A., et al. Desmin related disease: A matter of cell survival failure. Current Opinion in Cell Biology 2015;32:113-20. https://doi.org/10.1016/j.ceb.2015.01.004.

13. Goldfarb L.G., Olivé M., Vicart P., et al. Intermediate filament diseases: Desminopathy. Advances in Experimental Medicine and Biology 2008;642:131-64. https://doi.org/10.1007/978-0-387-84847-1_11.

14. Taylor M.R.G., Slavov D., Ku L., et al. Prevalence of desmin mutations in dilated cardiomyopathy. Circulation 2007;115:1244-51. https://doi.org/10.1161/CIRCULATIONAHA.106.646778.

15. Hunt S.A., Abraham W.T., Chin M.H., et al. ACC/AHA 2005 Guideline Update for the Diagnosis and Management of Chronic Heart Failure in the Adult. Circulation 2005;112. https://doi.org/10.1161/circulationaha.105.167586.

16. Heckmann M.B., Bauer R., Jungmann A., et al. AAV9-mediated gene transfer of desmin ameliorates cardiomyopathy in desmin-deficient mice. Gene Therapy 2016;23:673-9. https://doi.org/10.1038/gt.2016.40.

17. Wang X., Klevitsky R., Huang W., et al. αB-Crystallin Modulates Protein Aggregation of Abnormal Desmin. Circulation Research 2003;93:998-1005. https://doi.org/10.1161/01.RES.0000102401.77712.ED.

18. Pawelczak K.S., Gavande N.S., Vander Vere-Carozza P.S., et al. Modulating DNA Repair Pathways to Improve Precision Genome Engineering. ACS Chemical Biology 2018;13:389-96. https://doi.org/10.1021/acschembio.7b00777.

19. McLaughlin H.M., Kelly M.A., Hawley P.P., et al. Compound heterozygosity of predicted loss-of-function DES variants in a family with recessive desminopathy. BMC Medical Genetics 2013;14:68. https://doi.org/10.1186/1471-2350-14-68.

20. Brinkman E.K., Chen T., Amendola M., et al. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Research 2014;42. https://doi.org/10.1093/nar/gku936.