Circularization of genes and chromosome by CRISPR in human cells

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 McDonald Church; Lars Bolund; Birgitte Regenberg; Yonglun Luo

doi:10.1093/nar/gky767

GenomicsNucleic Acids ResearchAug 22, 2018

Peer-reviewed journal article

Circularization of genes and chromosome by CRISPR in human cells

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

Abstract

Extrachromosomal circular DNA (eccDNA) and ring chromosomes are genetic alterations found in humans with genetic disorders and diseases such as cancer. However, there is a lack of genetic engineering tool to recapitulate these features. Here, we report the discovery that delivery of pairs of CRISPR/Cas9 guide RNAs into human cells generate functional eccDNAs and ring chromosomes. We generated a dual-fluorescence eccDNA biosensor system, which allows us to study CRISPR deletion, inversion, and circularization of genes inside cells. Analysis after CRISPR editing at intergenic and genic loci in human embryonic kidney 293T cells and human mammary fibroblasts reveal that CRISPR deleted DNA readily form eccDNA in human cells. DNA in sizes from a few hundred base pairs up to a 47.4 megabase-sized ring chromosome (chr18) can be circularized. Our discoveries advance and expand CRISPR-Cas9 technology applications for genetic engineering, modeling of human diseases, and chromosome engineering.\n\nOne Sentence Summary: CRISPR circularization of DNA offers new tools for studying eccDNA biogenesis, function, chromosome engineering, and synthetic biology.

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