3
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing

      research-article

      Read this article at

      Bookmark
          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.

          Abstract

          Despite being time-consuming and costly, generating genome-edited pigs holds great promise for agricultural, biomedical, and pharmaceutical applications. To further facilitate genome editing in pigs, we report here establishment of a pig line with Cre-inducible Cas9 expression that allows a variety of ex vivo genome editing in fibroblast cells including single- and multigene modifications, chromosome rearrangements, and efficient in vivo genetic modifications. As a proof of principle, we were able to simultaneously inactivate five tumor suppressor genes ( TP53, PTEN, APC, BRCA1, and BRCA2) and activate one oncogene ( KRAS), achieved by delivering Cre recombinase and sgRNAs, which caused rapid lung tumor development. The efficient genome editing shown here demonstrates that these pigs can serve as a powerful tool for dissecting in vivo gene functions and biological processes in a temporal manner and for streamlining the production of genome-edited pigs for disease modeling.

          Related collections

          Most cited references51

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

          Multiplex genome engineering using CRISPR/Cas systems.

          Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            RNA-guided human genome engineering via Cas9.

            Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: found
              Is Open Access

              Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

              TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.
                Bookmark

                Author and article information

                Journal
                Genome Res
                Genome Res
                genome
                genome
                GENOME
                Genome Research
                Cold Spring Harbor Laboratory Press
                1088-9051
                1549-5469
                December 2017
                : 27
                : 12
                : 2061-2071
                Affiliations
                [1 ]CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China;
                [2 ]University of Chinese Academy of Sciences, Beijing 100049, China;
                [3 ]Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China;
                [4 ]Key Laboratory for Major Obstetric Diseases of Guangdong Province, Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, China;
                [5 ]Department of Nuclear Medicine, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou 510010, China;
                [6 ]Department of Cardiology, Experimental Cardiology Laboratory, University Medical Center Utrecht, Utrecht 3584CX, the Netherlands;
                [7 ]Netherlands Heart Institute, Utrecht 3584CX, the Netherlands;
                [8 ]Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus Hinxton, Cambridge CB10 1HH, United Kingdom;
                [9 ]Jilin Provincial Key Laboratory of Animal Embryo Engineering, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, China
                Author notes
                [10]

                These authors contributed equally to this work.

                Article
                9509184
                10.1101/gr.222521.117
                5741047
                29146772
                218aef1a-8f51-431c-bf4e-a3768b0749a3
                © 2017 Wang et al.; Published by Cold Spring Harbor Laboratory Press

                This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

                History
                : 9 March 2017
                : 26 October 2017
                Page count
                Pages: 11
                Funding
                Funded by: National Natural Science Foundation of China , open-funder-registry 10.13039/501100001809;
                Award ID: 81672317
                Award ID: 81671121
                Award ID: 31601187
                Award ID: 31401271
                Funded by: Bureau of International Cooperation, Chinese Academy of Sciences , open-funder-registry 10.13039/501100005200;
                Award ID: 154144KYSB20150033
                Funded by: Science and Technology Planning Project of Guangdong Province, China
                Award ID: 2013B060300017
                Award ID: 2014B020225003
                Award ID: 2014B030301058
                Award ID: 2015A030310119
                Award ID: 2016A020216023
                Award ID: 2016B030229006
                Funded by: Bureau of Science and Technology of Guangzhou Municipality
                Award ID: 201505011111498
                Categories
                Method

                Comments

                Comment on this article