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Review
. 2020 Feb;36(1):17-29.
doi: 10.1007/s10565-019-09488-2. Epub 2019 Aug 15.

Applying switchable Cas9 variants to in vivo gene editing for therapeutic applications

Emily M Mills  1 , Victoria L Barlow  1 , Louis Y P Luk  1 , Yu-Hsuan Tsai  2
Affiliations
Review

Applying switchable Cas9 variants to in vivo gene editing for therapeutic applications

Emily M Mills et al. Cell Biol Toxicol. 2020 Feb.

Abstract

Progress in targeted gene editing by programmable endonucleases has paved the way for their use in gene therapy. Particularly, Cas9 is an endonuclease with high activity and flexibility, rendering it an attractive option for therapeutic applications in clinical settings. Many disease-causing mutations could potentially be corrected by this versatile new technology. In addition, recently developed switchable Cas9 variants, whose activity can be controlled by an external stimulus, provide an extra level of spatiotemporal control on gene editing and are particularly desirable for certain applications. Here, we discuss the considerations and difficulties for implementing Cas9 to in vivo gene therapy. We put particular emphasis on how switchable Cas9 variants may resolve some of these barriers and advance gene therapy in the clinical setting.

Keywords: Endonuclease; Gene therapy; In vivo gene editing; Switchable Cas9.

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Figures

Fig. 1
Fig. 1
Two stages of precise gene editing involving a recognition of the target DNA by the endonuclease and subsequent cleavage to generate a double-strand break and b repair of the break by cellular mechanisms
Fig. 2
Fig. 2
Generation of a DNA double-strand break by Cas9 involving a formation of the ribonucleoprotein, b recognition of the target DNA, and c cleavage of the double-stranded DNA
Fig. 3
Fig. 3
Consequences of repairing a double-strand break by the two cellular mechanisms, a homology-directed repair (HDR) and b non-homologous end joining (NHEJ)
Fig. 4
Fig. 4
Exon skipping by NHEJ to restore an open reading frame. a Protein production from a normal or disease state DNA. b Exon skipping by cutting out the mutant exon. c Exon skipping by disrupting the intron-exon boundary
Fig. 5
Fig. 5
Regulation of SpCas9 by non-drug molecules. a Stability of the fusion protein containing SpCas9 and a FKBP12-derived destabilizing domain can be regulated by Shield-1 so that in the absence of Shield-1, all fusion proteins are rapidly degraded. b Genetic code expansion for site-specific non-canonical amino acid incorporation is used to control the production of full-length, functional SpCas9
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