CRISPR-C: 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 M. Church; Lars Bolund; Birgitte Regenberg; Yonglun Luo

doi:10.1093/nar/gky767

CRISPR-C: 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 M. Church, Lars Bolund, Birgitte Regenberg, Yonglun Luo

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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.

Original language	English
Article number	e131
Journal	Nucleic Acids Research
Volume	46
Issue number	22
Pages (from-to)	1-13
ISSN	0305-1048
DOIs	https://doi.org/10.1093/nar/gky767
Publication status	Published - 2018

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Møller, H. D., Lin, L., Xiang, X., Petersen, T. S., Huang, J., Yang, L., Kjeldsen, E., Jensen, U. B., Zhang, X., Liu, X., Xu, X., Wang, J., Yang, H., Church, G. M., Bolund, L., Regenberg, B., & Luo, Y. (2018). CRISPR-C: circularization of genes and chromosome by CRISPR in human cells. Nucleic Acids Research, 46(22), 1-13. Article e131. https://doi.org/10.1093/nar/gky767

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title = "CRISPR-C: circularization of genes and chromosome by CRISPR in human cells",

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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year = "2018",

doi = "10.1093/nar/gky767",

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T1 - CRISPR-C

T2 - circularization of genes and chromosome by CRISPR in human cells

AU - Møller, Henrik Devitt

AU - Lin, Lin

AU - Xiang, Xi

AU - Petersen, Trine Skov

AU - Huang, Jinrong

AU - Yang, Luhan

AU - Kjeldsen, Eigil

AU - Jensen, Uffe Birk

AU - Zhang, Xiuqing

AU - Liu, Xin

AU - Xu, Xun

AU - Wang, Jian

AU - Yang, Huanming

AU - Church, George M.

AU - Bolund, Lars

AU - Regenberg, Birgitte

AU - Luo, Yonglun

PY - 2018

Y1 - 2018

N2 - 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.

AB - 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.

U2 - 10.1093/nar/gky767

DO - 10.1093/nar/gky767

M3 - Journal article

C2 - 30551175

AN - SCOPUS:85058926466

SN - 0305-1048

VL - 46

SP - 1

EP - 13

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 22

M1 - e131

ER -

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

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