Genome Editing for the Study of Cardiovascular Diseases

Abstract

Purpose of review: The opportunities afforded through the recent advent of genome-editing technologies have allowed investigators to more easily study a number of diseases. The advantages and limitations of the most prominent genome-editing technologies are described in this review, along with potential applications specifically focused on cardiovascular diseases.

Recent findings: The recent genome-editing tools using programmable nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), have rapidly been adapted to manipulate genes in a variety of cellular and animal models. A number of recent cardiovascular disease-related publications report cases in which specific mutations are introduced into disease models for functional characterization and for testing of therapeutic strategies. Recent advances in genome-editing technologies offer new approaches to understand and treat diseases. Here, we discuss genome editing strategies to easily characterize naturally occurring mutations and offer strategies with potential clinical relevance.

Keywords: CRISPR/Cas9; Cardiovascular disease; Genome editing; TALEN; ZFN; iPSC.

Fig. 1

General overview of genome-editing tools. a The general architecture of zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) are shown here. For ZFNs, the DNA-binding zinc-finger domains (ZF) recognize 3–4 basepairs (bp) that account for a total recognition of a unique 9–18 bp DNA sequence. TALENs rely on components called TAL repeats that contain a highly conserved 33–35 amino acid repeat with differing, variable amino acids in the 12th and 13th position that can specifically recognize one of the four DNA bases. Both ZFNs and TALENs require a cleavage domain comprising the nuclease domain of the bacterial enzyme, FokI, that dimerizes upon DNA targeting. For the CRISPR/ Cas9 system, a Cas9 nuclease complexed with a synthetic “guide” RNA about 100 nucleotides in length can target and introduce mutations at a specific genomic site. The first 20 nucleotides or so of the guide RNA (protospacer) will recognize and hybridize to a complementary sequence on either stand of a DNA molecule immediately next to a protospacer-adjacent motif (PAM). All of these technologies introduce a double-strand break, as denoted by arrows. b When a DNA double-stranded break occurs, it is repaired by either non-homologous end-joining (NHEJ) or homology-directed repair (HDR). Since NHEJ is error-prone, repair can result in deletions or insertions (indels) at the break site, potentially resulting in frameshifts. During HDR, if a repair template is introduced that contains a mutation, that mutation will be permanently introduced into the genome upon repair