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Improving the specificity and efficacy of CRISPR/CAS9 and gRNA through target specific DNA reporter

Zhang, Jian-Hua, Pandey, Mritunjay, Kahler, John F., Loshakov, Anna, Harris, Benjamin, Dagur, Pradeep K., Mo, Yin-Yuan, Simonds, William F.
Journal of biotechnology 2014 v.189 pp. 1-8
DNA, DNA damage, DNA repair, RNA, chromatin, engineering, high-throughput nucleotide sequencing, screening
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.