CRISPR-Cas9 for in vivo Gene Therapy: Promise and Hurdles

Abstract

Owing to its easy-to-use and multiplexing nature, the genome editing tool CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease 9) is revolutionizing many areas of medical research and one of the most amazing areas is its gene therapy potentials. Previous explorations into the therapeutic potentials of CRISPR-Cas9 were mainly conducted in vitro or in animal germlines, the translatability of which, however, is either limited (to tissues with adult stem cells amenable to culture and manipulation) or currently impermissible (due to ethic concerns). Recently, important progresses have been made on this regard. Several studies have demonstrated the ability of CRISPR-Cas9 for in vivo gene therapy in adult rodent models of human genetic diseases delivered by methods that are potentially translatable to human use. Although these recent advances represent a significant step forward to the eventual application of CRISPR-Cas9 to the clinic, there are still many hurdles to overcome, such as the off-target effects of CRISPR-Cas9, efficacy of homology-directed repair, fitness of edited cells, immunogenicity of therapeutic CRISPR-Cas9 components, as well as efficiency, specificity, and translatability of in vivo delivery methods. In this article, we introduce the mechanisms and merits of CRISPR-Cas9 in genome editing, briefly retrospect the applications of CRISPR-Cas9 in gene therapy explorations and highlight recent advances, later we discuss in detail the challenges lying ahead in the way of its translatability, propose possible solutions, and future research directions.

Figure 1

Schematic representation of CRISPR-Cas9-mediated genome editing. (a) Schematic of CRISPR locus (from Streptococcus pyogenes). (b) Site-specific DNA cleavage by nuclease Cas9 directed by complementary between a single guide RNA (sgRNA) and the target sequence upon the presence of a protospacer-adjacent motif (PAM) on the opposite strand. (c) The resultant double-strand breaks (DSBs) are subsequently repaired either by nonhomologous end-joining (NHEJ) or by homology-directed repair (HDR) upon the existence of a donor template. NHEJ is more efficient than HDR but is error prone and may produce indel mutations, whereas HDR can provide a precise gene modification.