CRISPR-C: circularization of genes and chromosome by CRISPR in human cells
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CRISPR-C : circularization of genes and chromosome by CRISPR in human cells. / Møller, Henrik Devitt; Lin, Lin; Xiang, Xi; Petersen, Trine Skov; Huang, Jinrong; Yang, Luhan; Kjeldsen, Eigil; Jensen, Uffe Birk; Zhang, Xiuqing; Liu, Xin; Xu, Xun; Wang, Jian; Yang, Huanming; Church, George M.; Bolund, Lars; Regenberg, Birgitte; Luo, Yonglun.
In: Nucleic Acids Research, Vol. 46, No. 22, e131, 2018, p. 1-13.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
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
VL - 46
SP - 1
EP - 13
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 22
M1 - e131
ER -
ID: 211857959