Toward CRISPR Therapies for Cardiomyopathies

Takahiko Nishiyama, MD, PhD; Rhonda Bassel-Duby, PhD; Eric N. Olson, PhD


Circulation. 2021;144(18):1525-1527. 

In This Article

CRISPR/Cas9 Gene Editing

CRISPR (clustered regularly interspaced short palindromic repeats)–mediated genome editing involves 2 components, a single guide RNA complimentary to the target DNA sequence, and a CRISPR-associated endonuclease (eg, Cas9 [CRISPR-associated protein 9]).[1] DNA cleavage is induced by a Cas9–single-guide RNA ribonucleoprotein complex when the target DNA sequences pair with the single-guide RNA near a protospacer-adjacent motif (Figure). Repair of the double-stranded DNA break is mediated by nonhomologous end joining, which generates insertions or deletions, or by homology-directed repair, which precisely repairs double-stranded DNA breaks by insertion of a specific DNA sequence. Correction of genetic cardiomyopathies via gene editing would likely require nonhomologous end joining because the homology-directed repair machinery is absent in postmitotic cells.


Duchenne muscular dystrophy correction by CRISPR editing with double-stranded DNA breaks (A), base editing (B), and prime editing (C).
A, Nonhomologous end joining (NHEJ), which induces insertions or deletions (INDELS) at the cutting site, is the main mechanism for repair of double-stranded DNA breaks (DSB). Homologydirected repair (HDR) inserts a precise DNA fragment. NHEJ–mediated repair introduces INDELS to restore the open reading frame either by exon skipping or reframing in a deletion of Duchenne muscular dystrophy (DMD) at exon 44. B, Base editors convert A–T to G–C or C–G to T–A base pairs without double-stranded DNA breaks. This approach can be used to disrupt splice sites, thereby causing exon skipping, as shown for DMD at exon 52. C, Prime editing can introduce specific DNA sequences to reframe exons, as shown for DMD at exon 52. PAM indicates protospacer adjacent motif; pegRNA, prime-editing guide RNA; and sgRNA, single guide RNA.

Most mutations responsible for DMD involve exon deletions or duplications that disrupt the expression of the dystrophin protein, leading to progressive muscle degeneration and cardiomyopathy. CRISPR/Cas9 editing has been deployed in patient-derived induced pluripotent stem cells, as well as in mice and dogs with DMD, to restore dystrophin expression in cardiac and skeletal muscles.[2] For example, a deletion of exon 44 of the dystrophin gene generates a premature stop codon in exon 45, causing DMD, which can be corrected either by skipping or reframing of exon 45.