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Nucleic Acids Res. 2018 Dec 14;46(22):e131. doi: 10.1093/nar/gky767.

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

Møller HD1, Lin L2, Xiang X2,3,4, Petersen TS2, Huang J1,4,5,6, Yang L7, Kjeldsen E8, Jensen UB2,8, Zhang X3,5,6, Liu X4,5,6, Xu X4,5,6, Wang J4,5,6,9, Yang H4,5,6,9, Church GM7,10, Bolund L2,4,5,11, Regenberg B1, Luo Y2,4,5,6,11.

Author information

Department of Biology, Faculty of Science, University of Copenhagen, Denmark.
Department of Biomedicine, Aarhus University, Denmark.
BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, China.
BGI-Qingdao, Qingdao 266555, China.
BGI-Shenzhen, Shenzhen 518083, China.
China National GeneBank, BGI-Shenzhen, Shenzhen 518120, China.
eGenesis, Inc., Cambridge, MA 02139, USA.
Department of Clinical Medicine, Aarhus University, Denmark.
James D. Watson Institute of Genome Science, 310008 Hangzhou, China.
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, Qingdao 266555, China.


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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