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

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  • CRISPR-C

    Final published version, 8.47 MB, PDF document

  • Henrik Devitt Møller
  • Lin Lin
  • Xi Xiang
  • Trine Skov Petersen
  • Jinrong Huang
  • Luhan Yang
  • Eigil Kjeldsen
  • Uffe Birk Jensen
  • Xiuqing Zhang
  • Xin Liu
  • Xun Xu
  • Jian Wang
  • Huanming Yang
  • George M. Church
  • Lars Bolund
  • Regenberg, Birgitte
  • Yonglun Luo

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.

Original languageEnglish
Article numbere131
JournalNucleic Acids Research
Volume46
Issue number22
Pages (from-to)1-13
ISSN0305-1048
DOIs
Publication statusPublished - 2018

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