Improving the specificity and efficacy of CRISPR/CAS9 and gRNA through target specific DNA reporter
•A target DNA EGFP reporter for fast screening CRISPR/CAS9 and gRNA activities.•The reporter can be used to pre-screen the most specific CAS9 and gRNA pairs.•Identification of the N-terminal modification of CAS9 is critical for activities.•Double strand DNA break specificities are both CAS9 and gRNA dependent.
Genomic 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.
Journal: Journal of Biotechnology - Volume 189, 10 November 2014, Pages 1–8