title: Improving the specificity and efficacy of CRISPR/CAS9 and gRNA through target specific DNA reporter.
abstract: 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.
authors.affiliation: 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.
authors.forename: Jian-Hua
authors.initials: JH
authors.lastname: Zhang
authors.affiliation: Metabolic Diseases Branch, Bldg. 10, Room 8C-101, National Institute of Diabetes and Digestive and Kidney Diseases, United States.
authors.forename: Mritunjay
authors.initials: M
authors.lastname: Pandey
authors.affiliation: Metabolic Diseases Branch, Bldg. 10, Room 8C-101, National Institute of Diabetes and Digestive and Kidney Diseases, United States.
authors.forename: John F
authors.initials: JF
authors.lastname: Kahler
authors.affiliation: Metabolic Diseases Branch, Bldg. 10, Room 8C-101, National Institute of Diabetes and Digestive and Kidney Diseases, United States.
authors.forename: Anna
authors.initials: A
authors.lastname: Loshakov
authors.affiliation: Metabolic Diseases Branch, Bldg. 10, Room 8C-101, National Institute of Diabetes and Digestive and Kidney Diseases, United States.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Harris
authors.affiliation: Flow Cytometry Core Facility, Bldg. 10, Room 8C-104, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, United States.
authors.forename: Pradeep K
authors.initials: PK
authors.lastname: Dagur
authors.affiliation: Department of Pharmacology and Toxicology, Cancer Institute, The University of Mississippi Medical Center, 2500N State St., Room G651-3, Jackson, MS 39216, United States.
authors.forename: Yin-Yuan
authors.initials: YY
authors.lastname: Mo
authors.affiliation: Metabolic Diseases Branch, Bldg. 10, Room 8C-101, National Institute of Diabetes and Digestive and Kidney Diseases, United States.
authors.forename: William F
authors.initials: WF
authors.lastname: Simonds
mesh_terms: D000818:Animals; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA
keywords: CAS9; CRISPR; EGFP reporter; Specificity; gRNA