Single guide RNAs screening for restoring the reading frame of the DMD gene by genome editing

 Duchenne muscular dystrophy is the most common muscular dystrophy among children. Mostly the cause of the disease is frame-shift deletions of exons 43-55 of the DMD gene, leading to the absence of expression of the functional dystrophin. The most optimal approach
to restoring the reading frame is skipping an additional exon. In order to completely remove an exon, it is necessary to introduce two double-stranded DNA breaks, which is associated with a number of negative consequences. However, permanent exon skipping can
also be achieved by disruption the splice site by a single DNA break with following repair by non-homologous end joining. To assess the feasibility of this strategy, we performe a bioinformatics and experimental screening of sgRNAs targeting the splice sites of exons
43-55. As a result, the most promising sites for editing were the acceptor splice sites of exons 54-55 and the donor splice sites of exons 43 and 53. In addition, we demonstrate the efficiency of Cas9 with the non-classical PAM sequence NGA. 

1. Duan D., Goemans N., Takeda S. et al. Duchenne muscular dystrophy. Nat. Rev. Dis. Prim. England. 2021; 7(1): 13.

2. Bladen C.L., Salgado D., Monges S. et al. The TREAT-NMD DMD global database: Analysis of more than 7,000 duchenne muscular dystrophy mutations. Hum. Mutat. 2015;36(4):395-402.

3. Straub V., Guglieri M. An update on Becker muscular dystrophy. Curr. Opin. Neurol. England. 2023; 36(5): 450–454.

4. Chancellor D., Barrett D., Nguyen-Jatkoe L. et al. The state of cell and gene therapy in 2023. Mol. Ther. 2023; 31(12):3376-3388.

5. Hoy S.M. Delandistrogene Moxeparvovec: First Approval. Drugs. New Zealand. 2023; 83(14):1323-1329.

6. Monjaret F., Bourg N., Suel, L. et al. Cis-splicing and translation of the pre-trans-splicing molecule combine with efficiency in spliceosome-mediated RNA trans-splicing. Mol. Ther. 2014; 22(6):1176-1187.

7. Barthélémy F., Wein N., Krahn M. et al. Translational research and therapeutic perspectives in dysferlinopathies. Mol. Med. 2011; 17(9-10):875-882.

8. Athanasopoulos T., Munye M.M., Yáñez-Muñoz R.J. Nonintegrating Gene Therapy Vectors. Hematol Oncol Clin North Am. 2017; 31(5):753-770.

9. Izumi R., Takahashi T., Suzuki N. et al. The genetic profile of dysferlinopathy in a cohort of 209 cases: Genotype–phenotype relationship and a hotspot on the inner DysF domain. Hum. Mutat. 2020; 41(9):1540-1554.

10. Pierce E.A., Aleman T.S., Jayasundera K.T. et al. Gene Editing for CEP290-Associated Retinal Degeneration. N. Engl. J. Med. 2024; 390(21):1972–1984.

11. Gillmore J.D., Gane E., Taubel J. et al. CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis. N. Engl. J. Med. 2021; 385(6):493-502.

12. Hu J.H., Miller S.M., Geurts M.H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature. 2018; 556(7699): 57-63.

13. Sambrook J., Russell D.W. Preparation and Transformation of Competent E. coli Using Calcium Chloride. CSH Protoc. United States. 2006 Jun 1;2006(1):pdb.prot3932. doi: 10.1101/pdb.prot3932.

14. Hsu P.D., Scott D.A., Weinstein J.A. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. United States. 2013; 31(9): 827-832.

15. Doench J.G., Fusi N., Sullender M. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPRCas9. Nat. Biotechnol. United States. 2016; 34(2): 184-191.

16. Zhang Y., Ge X., Yang F. et al. Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells. Sci. ReС. 2014; 4: 1-5.

17. Wachutka L., Caizzi L., Gagneur J. et al. Global donor and acceptor splicing site kinetics in human cells. Elife. 2019; 8: 1-52.

18. Amoasii L., Long C., Li H. et al. Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy. Sci. Transl. Med. 2017; 9(418):eaan8081.

19. Kondratyeva E., Bukharova T., Efremova A. et al. Health Characteristics of Patients with Cystic Fibrosis whose Genotype Includes a Variant of the Nucleotide Sequence c.3140-16T>A and Functional Analysis of this Variant. Genes (Basel). 2021;12(6): 837.

20. Min Y.L., Chemello F., Li H. et al. Correction of Three Prominent Mutations in Mouse and Human Models of Duchenne Muscular Dystrophy by Single-Cut Genome Editing. Mol. Ther. 2020;28(9):2044-2055.