Stepping toward therapeutic CRISPR.

Most new technologies for manipulating gene expression in mammalian cells are accepted at a relatively slow pace. Occasionally, however, a new technology is so robust and fills such a critical niche that its adoption is widespread and rapid. Fifteen years ago, duplex RNAs were such a technology. RNA interference (RNAi) in mammalian cells was first demonstrated in 2001 (1) and within 2 y RNAi was a commonly used tool throughout industry and academia. RNAi is making its way into clinical trials as a potential therapeutic as challenges in delivery to relevant tissues begin to be overcome (2–4). More recently, another revolution in biology appears to be emerging, powered by bacterial type II clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated (Cas) systems. In PNAS, Rahdar et al. take a step toward a strategy that combines genetic and synthetic approaches for delivery of active CRISPR-Cas in vivo (5). CRISPR-Cas is based on a natural bacterial defense mechanism for controlling pathogens (6). The realization that CRISPR-Cas can efficiently direct cleavage of double-stranded DNA in diverse biological systems has rapidly transformed it into a deft tool for genome editing (7–9). The most popular CRISPR-Cas system makes use of the Cas9 endonuclease from Streptococcus pyogenes. Cas9 binds a short 42-nt-long CRISPR RNA (crRNA) and an 80-nt transactivating crRNA (tracrRNA). The crRNA has a variable guide sequence that directs Cas9 endonuclease activity to sequence-specifically cut both strands of a DNA target. Cleavage typically introduces insertions or deletions through errors in natural DNA repair mechanisms. The presence of an appropriate donor DNA can also result in accurate insertion of new sequences through homology-directed repair. By this method, permanent changes to the genome are accomplished. Thus, CRISPR-Cas represents a powerful research tool for understanding gene function (10). The widespread adoption of CRISPR-Cas provides objective evidence for its reliable use as a tool to investigate the basic biology of cellular processes. It is clear that, like duplex RNA for gene silencing, laboratory applications for CRISPR-Cas will proliferate (10–12). Moving beyond such applications, the potential for applying CRISPR-Cas to therapeutic development is less certain (13). Traditional small-molecule synthetic drugs are usually below 500 Da in molecular weight. In contrast, CRISPR-Cas is a large complex formed by a protein and two RNA molecules. Present technologies do not offer straightforward solutions for direct entry of such complexes into tissues and cells if administered to patients. Antibodies form an increasingly successful class of drugs, but this success has been facilitated by the fact that, unlike CRISPRCas, they function by binding targets outside of cells and do not need to be internalized. One option would be to use gene therapy to introduce vectors designed to express the endonuclease domain and a fusion of the crRNA and tracrRNA domains, called a single guide RNA (sgRNA) (13). One problem for solely relying on gene therapy to deliver CRISPR components is that, unlike synthetic drugs, where administration can be stopped, once expressed inside cells a fully functional CRISPR-Cas complex might be difficult to turn off, especially if the Cas9 and sgRNA genes are incorporated into the genome. Gene therapy to deliver all CRISPR components may ultimately prove to be a successful