Circularization of genes and chromosome by CRISPR in human cells
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 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. One Sentence Summary: CRISPR circularization of DNA offers new tools for studying eccDNA biogenesis, function, chromosome engineering, and synthetic biology.
Lin Lin、Devitt M?ller Henrik、Xu Xun、Regenberg Birgitte、Luo Yonglun、Liu Xin、Xiang Xi、Petersen Trine Skov、Zhang Xiuqing、Jensen Uffe Birk、Yang Luhan、Church George M.、Wang Jian、Bolund Lars、Huang Jinrong、Yang Huanming、Kjeldsen Eigil
Department of Biomedicine, Aarhus UniversityDepartment of Biology, Faculty of Science, University of CopenhagenBGI-Shenzhen||BGI-Qingdao||China National GeneBank, BGI-ShenzhenDepartment of Biology, Faculty of Science, University of CopenhagenDepartment of Biomedicine, Aarhus University||BGI-Shenzhen||BGI-Qingdao||China National GeneBank, BGI-Shenzhen||Lars Bolund Institute of Regenerative Medicine, BGI-QingdaoBGI-Shenzhen||BGI-Qingdao||China National GeneBank, BGI-ShenzhenBGI Education Center, University of Chinese Academy of Sciences||Department of Biomedicine, Aarhus University||BGI-QingdaoDepartment of Biomedicine, Aarhus UniversityBGI Education Center, University of Chinese Academy of Sciences||BGI-Shenzhen||China National GeneBank, BGI-ShenzhenDepartment of Biomedicine, Aarhus University||Department of Clinical Medicine, Aarhus UniversityeGenesis, Inc.eGenesis, Inc.||Department of Genetics, Harvard Medical SchoolBGI-Shenzhen||BGI-Qingdao||China National GeneBank, BGI-ShenzhenDepartment of Biomedicine, Aarhus University||BGI-Shenzhen||BGI-Qingdao||Lars Bolund Institute of Regenerative Medicine, BGI-QingdaoDepartment of Biology, Faculty of Science, University of Copenhagen||BGI-Shenzhen||BGI-Qingdao||China National GeneBank, BGI-ShenzhenBGI-Shenzhen||BGI-Qingdao||China National GeneBank, BGI-ShenzhenDepartment of Clinical Medicine, Aarhus University
遗传学分子生物学生物工程学
Lin Lin,Devitt M?ller Henrik,Xu Xun,Regenberg Birgitte,Luo Yonglun,Liu Xin,Xiang Xi,Petersen Trine Skov,Zhang Xiuqing,Jensen Uffe Birk,Yang Luhan,Church George M.,Wang Jian,Bolund Lars,Huang Jinrong,Yang Huanming,Kjeldsen Eigil.Circularization of genes and chromosome by CRISPR in human cells[EB/OL].(2025-03-28)[2026-08-26].https://www.biorxiv.org/content/10.1101/304493.点此复制
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