Genome Editing and Cardiac Arrhythmias

Figures

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  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,…

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  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.