Delivering the second revolution in site-specific nucleases

Abstract

Viruses have been used to deliver two types of site-specific nucleases into cells for targeted gene editing.

Keywords: gag; gene therapy; lentiviral vector; protein transduction; transcription activator-like effector nucleases; zinc-finger nucleases.

Figure 1.. Modified viruses can deliver the enzymes and templates needed for gene editing.

The lentiviral particles created by Cai et al. had glycoproteins (VSV; pale orange) on their surface, which enabled them to infect of a wide range of cell types. The lentiviral particles also contained site-specific nucleases (thick black lines) that targeted specific sequences in the DNA of the host cell, and a DNA template (thick red line) that was added to the genome of the host cell. Cai et al. demonstrated that their approach worked with two types of site-specific nuclease (SSN): zinc finger nucleases (ZFNs) and transcription activator-like endonucleases (TALENs). The modified lentiviral ‘genome’ is illustrated at the top: when this is expressed as a polypeptide inside the host cell, it is cleaved by protease (PROT; red stars). The gene for the SSN was inserted into the gag sequence of genes in the virus. Cai et al. also made a number of other important modifications: the normal lentiviral promoter that marks the start of a gene was replaced by a different promoter (called CMV), and the gene in the pol sequence that encodes an enzyme called integrase (INT) was modified (red slash) to inactivate the integration activity. The virus used was actually an RNA virus, so reverse transcriptase (RT; brown stars) was used to convert the RNA into DNA. The bottom of the figure shows the DNA of the host cell. The SSNs bind to the DNA (double blue lines) at the sites indicated by the red stars, and the cleavage site is shown by the lightning bolt. VSV: vesicular stomatitis virus; CMV: cytomegalovirus.