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      CRISPR-C: circularization of genes and chromosome by CRISPR in human cells

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          Abstract

          Extrachromosomal circular DNA (eccDNA) and ring chromosomes are genetic alterations found in humans with genetic disorders. However, there is a lack of genetic engineering tools to recapitulate and study the biogenesis of eccDNAs. Here, we created a dual-fluorescence biosensor cassette, which upon the delivery of pairs of CRISPR/Cas9 guide RNAs, CRISPR-C, allows us to study the biogenesis of a specific fluorophore expressing eccDNA in human cells. We show that CRISPR-C can generate functional eccDNA, using the novel eccDNA biosensor system. We further reveal that CRISPR-C also can generate eccDNAs from intergenic and genic loci in human embryonic kidney 293T cells and human mammary fibroblasts. EccDNAs mainly forms by end-joining mediated DNA-repair and we show that CRISPR-C is able to generate endogenous eccDNAs in sizes from a few hundred base pairs and ranging up to 207 kb. Even a 47.4 megabase-sized ring chromosome 18 can be created by CRISPR-C. Our study creates a new territory for CRISPR gene editing and highlights CRISPR-C as a useful tool for studying the cellular impact, persistence and function of eccDNAs.

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          Identification of genes that are associated with DNA repeats in prokaryotes.

          Ruud Jansen, Jan Embden, Wim Gaastra … (2002)
          Using in silico analysis we studied a novel family of repetitive DNA sequences that is present among both domains of the prokaryotes (Archaea and Bacteria), but absent from eukaryotes or viruses. This family is characterized by direct repeats, varying in size from 21 to 37 bp, interspaced by similarly sized non-repetitive sequences. To appreciate their characteri-stic structure, we will refer to this family as the clustered regularly interspaced short palindromic repeats (CRISPR). In most species with two or more CRISPR loci, these loci were flanked on one side by a common leader sequence of 300-500 b. The direct repeats and the leader sequences were conserved within a species, but dissimilar between species. The presence of multiple chromosomal CRISPR loci suggests that CRISPRs are mobile elements. Four CRISPR-associated (cas) genes were identified in CRISPR-containing prokaryotes that were absent from CRISPR-negative prokaryotes. The cas genes were invariably located adjacent to a CRISPR locus, indicating that the cas genes and CRISPR loci have a functional relationship. The cas3 gene showed motifs characteristic for helicases of the superfamily 2, and the cas4 gene showed motifs of the RecB family of exonucleases, suggesting that these genes are involved in DNA metabolism or gene expression. The spatial coherence of CRISPR and cas genes may stimulate new research on the genesis and biological role of these repeats and genes.
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            Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9

            Dong Niu, Hong-Jiang Wei, Lin Lin … (2017)
            Xenotransplantation is a promising strategy to alleviate the shortage of organs for human transplantation. In addition to the concern on pig-to-human immunological compatibility, the risk of cross-species transmission of porcine endogenous retroviruses (PERVs) has impeded the clinical application of this approach. Earlier, we demonstrated the feasibility of inactivating PERV activity in an immortalized pig cell line. Here, we confirmed that PERVs infect human cells, and observed the horizontal transfer of PERVs among human cells. Using CRISPR-Cas9, we inactivated all the PERVs in a porcine primary cell line and generated PERV-inactivated pigs via somatic cell nuclear transfer. Our study highlighted the value of PERV inactivation to prevent cross-species viral transmission and demonstrated the successful production of PERV-inactivated animals to address the safety concern in clinical xenotransplantation.
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              Characterization of genomic deletion efficiency mediated by clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells.

              Matthew Canver, Daniel E Bauer, Abhishek Dass … (2014)
              The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Nucleic Acids Res
                Journal ID (iso-abbrev): Nucleic Acids Res
                Journal ID (publisher-id): nar
                Title: Nucleic Acids Research
                Publisher: Oxford University Press
                ISSN (Print): 0305-1048
                ISSN (Electronic): 1362-4962
                Publication date (Print): 14 December 2018
                Publication date (Electronic): 24 August 2018
                Publication date PMC-release: 24 August 2018
                Volume: 46
                Issue: 22
                Page: e131
                Affiliations
                [1 ]Department of Biology, Faculty of Science, University of Copenhagen, Denmark
                [2 ]Department of Biomedicine, Aarhus University, Denmark
                [3 ]BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, China
                [4 ]BGI-Qingdao, Qingdao 266555, China
                [5 ]BGI-Shenzhen, Shenzhen 518083, China
                [6 ]China National GeneBank, BGI-Shenzhen, Shenzhen 518120, China
                [7 ]eGenesis, Inc., Cambridge, MA 02139, USA
                [8 ]Department of Clinical Medicine, Aarhus University, Denmark
                [9 ]James D. Watson Institute of Genome Science, 310008 Hangzhou, China
                [10 ]Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
                [11 ]Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, Qingdao 266555, China
                Author notes
                To whom correspondence should be addressed. Tel: +45 87 16 77 61; Fax: +45 87 16 77 61; Email: alun@ 123456biomed.au.dk . Correspondence may also be addressed to Birgitte Regenberg. Tel: +45 35 32 16 80; Fax: +45 35 32 16 80; Email: bregenberg@ 123456bio.ku.dk

                The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.

                Lead contact.

                Author information
                http://orcid.org/0000-0002-0007-7759
                Article
                Publisher ID: gky767
                DOI: 10.1093/nar/gky767
                PMC ID: 6294522
                PubMed ID: 30551175
                SO-VID: 24100994-3be5-443d-909d-085bb0df9c22
                Copyright © © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date accepted : 20 August 2018
                Date revision received : 28 July 2018
                Date received : 11 May 2018
                Page count
                Pages: 13
                Funding
                Funded by: Danish Council for Independent Research 10.13039/501100004836
                Award ID: FNU 6108-00171B
                Funded by: Carlsberg foundation 10.13039/501100002808
                Award ID: CF17-0226
                Funded by: Lundbeck Foundation 10.13039/501100003554
                Award ID: R219-2016-1375
                Funded by: Danish Research Council for Independent Research 10.13039/501100004836
                Award ID: DFF-1337-00128
                Funded by: Sapere Aude Young Research Talent Prize
                Award ID: DFF-1335-00763A
                Funded by: Lundbeck Foundation 10.13039/501100003554
                Award ID: R173-2014-1105
                Categories
                Subject: Methods Online

                ScienceOpen disciplines: Genetics
                Data availability:
                ScienceOpen disciplines: Genetics

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