Molecular basis, applications and challenges of CRISPR/Cas9: a continuously evolving tool for genome editing.

The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system is a recently discovered tool for genome editing that has quickly revolutionized the ability to generate site-specific mutations in a wide range of animal models, including nonhuman primates. Indeed, a significant number of scientific reports describing single or multiplex guide RNA microinjection, double-nicking strategies, site-specific knock-in and conditional knock-out have been published in less than three years. However, despite the great potential of this new technology, there are some limitations because of the presence of off-target genomic sites, which must be taken into consideration. To address this issue, various research teams have tried to improve the efficiency of the system through enzymatic modifications of the Cas9 protein or by the introduction of alternative strategies. Although several review articles are available that singly describe the molecular mechanism(s), applications and challenges of each of these strategies, a concise compilation of approaches is lacking. In the current review, we describe and evaluate most CRISPR/Cas9 approaches available at present, describing both mechanism of action, in addition to advantages or disadvantages. The primary goal of this work is to serve as a guide for not skilled researchers, facilitating the selection of the best strategy to target their gene of interest and allowing optimization of particular applications to the specific aims of the study. The present article also offers a unique perspective, focusing on the fact that CRISPR technology is opening a new genomic era, providing the means to manipulate specific genes in a targeted manner in all animal models, an endeavor previously considered to be difficult.