Genome Editing and Cardiac Arrhythmias

Abstract

This article reviews progress in the field of cardiac genome editing, in particular, its potential utility in treating cardiac arrhythmias. First, we discuss genome editing methods by which DNA can be disrupted, inserted, deleted, or corrected in cardiomyocytes. Second, we provide an overview of in vivo genome editing in preclinical models of heritable and acquired arrhythmias. Third, we discuss recent advancements in cardiac gene transfer, including delivery methods, gene expression optimization, and potential adverse effects associated with therapeutic somatic genome editing. While genome editing for cardiac arrhythmias is still in its infancy, this approach holds great promise, especially for inherited arrhythmia syndromes with a defined genetic defect.

Keywords: CRISPR/Cas9; RyR2; arrhythmias; catecholaminergic polymorphic ventricular tachycardia; genome editing; long QT syndrome; ryanodine receptor.

Figure 1

Schematic overview of major CRISPR/Cas9-based genome editing technologies. (A) Conventional CRISPR/Cas9 editing of a mutant allele involves selecting guide RNAs adjacent to a protospacer adjacent motif (PAM) site, with a mutation in the spacer sequence (14–19) to minimize wild-type allele cleavage. The Cas9 endonuclease introduces a double-stranded break (DSB). Non-homologous end joining (NHEJ) repairs the mutant allele but introduces insertions and deletions at the mutation site, which can lead to frame-shift mutations and stop codons, resulting in nonsense-mediated decay of mutant RNA (top). On the other hand, homology-directed repair (HDR) is less efficient but leads to correction of the mutant allele using a DNA repair template. (B) Cytosine base editors are created by fusing Cas9 nickase (nCas9) or catalytically inactive “dead” Cas9 (dCas9) to a cytidine deaminase. Base editors are targeted to a specific locus using gRNA. They can convert cytidine (C) to uridine (U) within a small editing window (4–7 in the spacer sequence, depending on the editor type), near the PAM site. Uridine is subsequently converted to thymidine (T) through base excision repair. (C) Likewise, adenosine base editors have been engineered to convert adenosine (A) to inosine (I), which is subsequently converted to guanidine (G). (D) Prime editing involves a reverse transcriptase and dCas9 that can produce single-stranded DNA breaks. The 3′-extended single guide RNA contains a primer binding site and a reverse transcriptase template, which is referred to as primer editing guide RNA (pegRNA). Hybridization of the exposed 3′-end to the primer binding site primes the reverse transcription of the nicked DNA strand for the desired edit.

Figure 2

Schematic overview of therapeutic CRISPR/Cas9 targets for arrhythmia treatment. The diagram shows the plasma membrane with various ion channels including the voltage-gated L-type Ca²⁺ channel (LTCC), and K⁺ and Na⁺ channels. The sarcoplasmic reticulum (SR) is shown with the ryanodine receptor type-2 (RyR2)/Ca²⁺ release channel and sarco-/endoplasmic Ca²⁺-ATPase (SERCA2a) with its regulatory subunit phospholamban (PLN). Dystrophin links the plasmalemmal sarcoglycan complex to the sarcomere. The Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) phosphorylates various ion channels and Ca²⁺ handling proteins within cardiomyocytes. Gene names that are targets of therapeutic genome editing are indicated in bold and italicized.