+1 Recommend
0 collections
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Off-target Effects in CRISPR/Cas9-mediated Genome Engineering


      Read this article at

          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.


          CRISPR/Cas9 is a versatile genome-editing technology that is widely used for studying the functionality of genetic elements, creating genetically modified organisms as well as preclinical research of genetic disorders. However, the high frequency of off-target activity (≥50%)—RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site—is one major concern, especially for therapeutic and clinical applications. Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for minimizing off-target cleavage. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype–phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology.

          Related collections

          Most cited references24

          • Record: found
          • Abstract: found
          • Article: not found

          CRISPR RNA-guided activation of endogenous human genes

          Catalytically inactive CRISPR-associated 9 nuclease (dCas9) can be directed by short guide RNAs (gRNAs) to repress endogenous genes in bacteria and human cells. Here we show that a dCas9-VP64 transcriptional activation domain fusion protein can be directed by single or multiple gRNAs to increase expression of specific endogenous human genes. These results provide an important proof-of-principle that CRISPR-Cas systems can be used to target heterologous effector domains in human cells.
            • Record: found
            • Abstract: found
            • Article: not found

            Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects.

            Bacterial RNA-directed Cas9 endonuclease is a versatile tool for site-specific genome modification in eukaryotes. Co-microinjection of mouse embryos with Cas9 mRNA and single guide RNAs induces on-target and off-target mutations that are transmissible to offspring. However, Cas9 nickase can be used to efficiently mutate genes without detectable damage at known off-target sites. This method is applicable for genome editing of any model organism and minimizes confounding problems of off-target mutations.
              • Record: found
              • Abstract: found
              • Article: not found

              Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease.

              RNA-guided genome editing with the CRISPR-Cas9 system has great potential for basic and clinical research, but the determinants of targeting specificity and the extent of off-target cleavage remain insufficiently understood. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) in HEK293T cells, in combination with 12 different single guide RNAs (sgRNAs). The number of off-target sites bound by dCas9 varied from ∼10 to >1,000 depending on the sgRNA. Analysis of off-target binding sites showed the importance of the PAM-proximal region of the sgRNA guiding sequence and that dCas9 binding sites are enriched in open chromatin regions. When targeted with catalytically active Cas9, some off-target binding sites had indels above background levels in a region around the ChIP-seq peak, but generally at lower rates than the on-target sites. Our results elucidate major determinants of Cas9 targeting, and we show that ChIP-seq allows unbiased detection of Cas9 binding sites genome-wide.

                Author and article information

                Mol Ther Nucleic Acids
                Mol Ther Nucleic Acids
                Molecular Therapy. Nucleic Acids
                Nature Publishing Group
                November 2015
                17 November 2015
                1 November 2015
                : 4
                : 11
                : e264
                [1 ]Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases, College of Veterinary Medicine, South China Agricultural University , Guangzhou, China
                [2 ]Faculty of Life Science and Technology, Kunming University of Science and Technology , Kunming, China
                [3 ]McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology , Cambridge, Massachusetts, USA
                [4 ]Department of Cell Biology & Institute of Biomedicine, College of Life Science and Technology, Jinan University , Guangzhou, China
                Author notes
                [* ]Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China. E-mail: yangsh@ 123456scau.edu.cn
                Copyright © 2015 Official journal of the American Society of Gene & Cell Therapy

                This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/4.0/

                : 31 July 2015
                : 05 October 2015

                Molecular medicine
                Molecular medicine
                crispr/cas9, off-target


                Comment on this article