title: 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.
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, Beijing, China.|Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China.
authors.forename: Rongxue
authors.initials: R
authors.lastname: Peng
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, Beijing, China.
authors.forename: Guigao
authors.initials: G
authors.lastname: Lin
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, Beijing, China.
authors.forename: Jinming
authors.initials: J
authors.lastname: Li
mesh_terms: D001665:Binding Sites / Q000235:genetics; D064113:CRISPR-Cas Systems; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods*; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D008957:Models, Genetic; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D015203:Reproducibility of Results
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: CRISPR/Cas systems; Cas9; DNA cleavage; gene targeting; genome editing; potential pitfalls; sgRNA; target specificity