Potential pitfalls of CRISPR/Cas9‐mediated genome editing

Abstract

Recently, a novel technique named the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein (Cas)9 system has been rapidly developed. This genome editing tool has improved our ability tremendously with respect to exploring the pathogenesis of diseases and correcting disease mutations, as well as phenotypes. With a short guide RNA, Cas9 can be precisely directed to target sites, and functions as an endonuclease to efficiently produce breaks in DNA double strands. Over the past 30 years, CRISPR has evolved from the ‘curious sequences of unknown biological function’ into a promising genome editing tool. As a result of the incessant development in the CRISPR/Cas9 system, Cas9 co‐expressed with custom guide RNAs has been successfully used in a variety of cells and organisms. This genome editing technology can also be applied to synthetic biology, functional genomic screening, transcriptional modulation and gene therapy. However, although CRISPR/Cas9 has a broad range of action in science, there are several aspects that affect its efficiency and specificity, including Cas9 activity, target site selection and short guide RNA design, delivery methods, off‐target effects and the incidence of homology‐directed repair. In the present review, we highlight the factors that affect the utilization of CRISPR/Cas9, as well as possible strategies for handling any problems. Addressing these issues will allow us to take better advantage of this technique. In addition, we also review the history and rapid development of the CRISPR/Cas system from the time of its initial discovery in 2012.

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

“…For demonstration of the functionality of the CRISPR-Cas9 system in B. subtilis, two deletions were introduced into the genome, a very large deletion with 25.1 kb containing the amyE gene and a shorter deletion with 4.1 kb containing the pulcherrimin biosynthesis genes. In the meantime, a range of sequence features in and around the target sequences that predict sgRNA efficiency have been identified (37). With the help of the sgRNA Designer tool provided by the Broad Institute (38), candidate sgRNA sequences were identified in the amyE sequence, and a high-scoring sequence was chosen for oligonucleotide synthesis.…”

The future of iPS cells in advancing regenerative medicine

“…These pitfalls have been extensively discussed elsewhere (Peng et al, 2015). Generally, the off-target effect is the major concern.…”

Excitation et crispations autour de CRISPR : lorsque la réalité dépasse la science-fiction

“…C'est pour cela que, dans le domaine de la myologie, les publications rapportant une édition efficace du gène de la dystrophine (DMD) [27][28][29][30] ne sont que des POC expérimentales et non des avancées thérapeutiques immédiatement applicables en clinique 63 . Les avantages de l'édition génomique que sont la correction in situ (donc régulée) et définitive sont contrebalancés par la nécessité absolue de réussir sans bavure (aucun impact off-target) et le risque que comporte l'abandon du bistouri (l'endonucléase Cas9) dans le champ opératoire 64 Or, les méthodes d'édition génomique en vigueur, même très améliorées, sont encore loin d'offrir les garanties de sécurité nécessaires 65,66 . Le bon sens nous amène à peser soigneusement les maladies qui pourraient être candidates à une éditothérapie.…”