A variant CRISPR-Cas9 system adds versatility to genome engineering

Targeted genome engineering has been instrumental for the study of biological processes, and it holds great promise for the treatment of disease. Historically, gene targeting by homologous recombination has been the preferred method to modify specific genes in mouse and human cells (Fig. 1). However, this approach is hampered by low efficiency, the requirement for drug selection to detect targeted cells, and the limited number of cell types and organisms amenable to the method. To overcome these limitations, technologies based on sequence-specific zinc finger (ZF) and transcription activator-like effector (TALE) proteins have been developed. These proteins can be engineered to theoretically recognize any DNA sequence in the genome. When fused to a nuclease domain and assembled in pairs flanking a target site of interest, ZF and TALE nucleases (ZFNs and TALENs) will introduce double-strand breaks (DSBs) on DNA. DSBs serve as substrates for nonhomologous end joining (NHEJ) or homology-directed repair (HDR), which in turn facilitate the engineering of targeted mutations, repair of endogenous mutations, or introduction of transgenic DNA elements. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR-associated (Cas) system represents the latest addition to this arsenal of tools for site-specific genome engineering (see below). Although each of these three gene modification approaches has advantages and disadvantages (Fig. 1), the pace and ease with which the CRISPR-Cas systems have been adapted to modify genes in different cell types and organisms suggests that it may very well become the new method of choice for genome engineering. In PNAS, Hou et al. introduce a unique variant of the Cas9 enzyme (1), which provides additional flexibility and specificity to the CRISPR system and to genome-modifying tools in general.