• Jian-Hua Zhang, Mritunjay Pandey, John F Kahler, Anna Loshakov, Benjamin Harris, Pradeep K Dagur, Yin-Yuan Mo, and William F Simonds.
• Metabolic Diseases Branch, Bldg. 10, Room 8C-101, National Institute of Diabetes and Digestive and Kidney Diseases, United States. Electronic address: jianhuaz@mail.nih.gov.
• J. Biotechnol. 2014 Nov 10; 189: 1-8.

AbstractGenomic engineering by the guide RNA (gRNA)-directed CRISPR/CAS9 is rapidly becoming a method of choice for various biological systems. However, pressing concerns remain regarding its off-target activities and wide variations in efficacies. While next generation sequencing (NGS) has been primarily used to evaluate the efficacies and off-target activities of gRNAs, it only detects the imperfectly repaired double strand DNA breaks (DSB) by the error-prone non-homologous end joining (NHEJ) mechanism and may not faithfully represent the DSB activities because the efficiency of NHEJ-mediated repair varies depending on the local chromatin environment. Here we describe a reporter system for unbiased detection and comparison of DSB activities that promises to improve the chance of success in genomic engineering and to facilitate large-scale screening of CAS9 activities and gRNA libraries. Additionally, we demonstrated that the tolerances to mismatches between a gRNA and the corresponding target DNA can occur at any position of the gRNA, and depend on both specific gRNA sequences and CAS9 constructs used. Published by Elsevier B.V.

12 keywords...

hide…