Comprehensive transcriptome-wide analysis of spliceopathy correction of myotonic dystrophy using CRISPR-Cas9 in iPSCs-derived cardiomyocytes

CTG repeat expansion (CTGexp) is associated with aberrant alternate splicing that contributes to cardiac dysfunction in myotonic dystrophy type 1 (DM1). Excision of this CTGexp repeat using CRISPR-Cas resulted in the disappearance of punctate ribonuclear foci in cardiomyocyte-like cells derived from...

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Bibliographic Details
Published in:Molecular therapy 2022-01, Vol.30 (1), p.75-91
Main Authors: Dastidar, Sumitava, Majumdar, Debanjana, Tipanee, Jaitip, Singh, Kshitiz, Klein, Arnaud F., Furling, Denis, Chuah, Marinee K., VandenDriessche, Thierry
Format: Article
Language:English
Subjects:
alternate splicing
Alternative Splicing
Biochemistry, Molecular Biology
Calmodulin-Binding Proteins - genetics
Cardiology and cardiovascular system
cardiomyogenic differentiation
CRISPR ribonucleoprotein
CRISPR-Cas Systems
gene editing
Genomics
Human health and pathology
Humans
induced pluripotent stem cells
Induced Pluripotent Stem Cells - metabolism
Life Sciences
Myocytes, Cardiac - metabolism
myotonic dystrophy
Myotonic Dystrophy - genetics
Myotonic Dystrophy - therapy
Myotonin-Protein Kinase - genetics
Original
ribonuclear foci
RNA-Binding Proteins - genetics
RNA-Binding Proteins - metabolism
spliceopathy
Transcriptome
Trinucleotide Repeat Expansion - genetics
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Summary:CTG repeat expansion (CTGexp) is associated with aberrant alternate splicing that contributes to cardiac dysfunction in myotonic dystrophy type 1 (DM1). Excision of this CTGexp repeat using CRISPR-Cas resulted in the disappearance of punctate ribonuclear foci in cardiomyocyte-like cells derived from DM1-induced pluripotent stem cells (iPSCs). This was associated with correction of the underlying spliceopathy as determined by RNA sequencing and alternate splicing analysis. Certain genes were of particular interest due to their role in cardiac development, maturation, and function (TPM4, CYP2J2, DMD, MBNL3, CACNA1H, ROCK2, ACTB) or their association with splicing (SMN2, GCFC2, MBNL3). Moreover, while comparing isogenic CRISPR-Cas9-corrected versus non-corrected DM1 cardiomyocytes, a prominent difference in the splicing pattern for a number of candidate genes was apparent pertaining to genes that are associated with cardiac function (TNNT, TNNT2, TTN, TPM1, SYNE1, CACNA1A, MTMR1, NEBL, TPM1), cellular signaling (NCOR2, CLIP1, LRRFIP2, CLASP1, CAMK2G), and other DM1-related genes (i.e., NUMA1, MBNL2, LDB3) in addition to the disease-causing DMPK gene itself. Subsequent validation using a selected gene subset, including MBNL1, MBNL2, INSR, ADD3, and CRTC2, further confirmed correction of the spliceopathy following CTGexp repeat excision. To our knowledge, the present study provides the first comprehensive unbiased transcriptome-wide analysis of the differential splicing landscape in DM1 patient-derived cardiac cells after excision of the CTGexp repeat using CRISPR-Cas9, showing reversal of the abnormal cardiac spliceopathy in DM1. [Display omitted] DM1 is due to pathogenic CTG repeats in the DMPK gene that trigger spliceopathy and cardiac dysfunction. Comprehensive transcriptome-wide analysis shows that CRISPR-Cas9-mediated excision of the CTG repeats reverses spliceopathy in DM1 patient-derived cardiomyocyte-like cells. These corrected versus non-corrected isogenic lines provide new molecular insights in DM1 pathology.
ISSN:1525-0016
1525-0024
DOI:10.1016/j.ymthe.2021.08.004