CRISPR/Cas9-mediated efficient and precise targeted integration of donor DNA harboring double cleavage sites in Xenopus tropicalis

Cheng Zhou Mao, Li Zheng, Yi Min Zhou, Hai Yan Wu, Jing Bo Xia, Chi Qian Liang, Xiao Fang Guo, Wen Tao Peng, Hui Zhao, Wei Bin Cai, Soo Ki Kim, Kyu Sang Park, Dong Qing Cai, Xu Feng Qi

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system has emerged as a powerful tool for knock-in of DNA fragments via donor plasmid and homology-independent DNA repair mechanism; however, conventional integration includes unnecessary plasmid backbone and may result in the unfaithful expression of the modified endogenous genes. Here, we report an efficient and precise CRISPR/Cas9-mediated integration strategy using a donor plasmid that harbors 2 of the same cleavage sites that flank the cassette at both sides. After the delivery of donor plasmid, together with Cas9 mRNA and guide RNA, into cells or fertilized eggs, concurrent cleavages at both sides of the exogenous cassette and the desired chromosomal site result in precise targeted integration without plasmid backbone. We successfully used this approach to precisely integrate the EGFP reporter gene into the myh6 locus or the GAPDH locus in Xenopus tropicalis or human cells, respectively. Furthermore, we demonstrate that replacing conventional terminators with the endogenous 3UTR of target genes in the cassette greatly improves the expression of reporter gene after integration. Our efficient and precise method will be useful for a variety of targeted genome modifications, not only in X. tropicalis, but also in mammalian cells, and can be readily adapted to many other organisms.

Original languageEnglish
Pages (from-to)6495-6509
Number of pages15
JournalFASEB Journal
Volume32
Issue number12
DOIs
Publication statusPublished - 2018 Dec

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Xenopus
Plasmids
Genes
DNA
Reporter Genes
Guide RNA
Cells
Zygote
Ports and harbors
DNA Repair
Repair
Genome
Messenger RNA

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

Cite this

Mao, Cheng Zhou ; Zheng, Li ; Zhou, Yi Min ; Wu, Hai Yan ; Xia, Jing Bo ; Liang, Chi Qian ; Guo, Xiao Fang ; Peng, Wen Tao ; Zhao, Hui ; Cai, Wei Bin ; Kim, Soo Ki ; Park, Kyu Sang ; Cai, Dong Qing ; Qi, Xu Feng. / CRISPR/Cas9-mediated efficient and precise targeted integration of donor DNA harboring double cleavage sites in Xenopus tropicalis. In: FASEB Journal. 2018 ; Vol. 32, No. 12. pp. 6495-6509.
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abstract = "The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system has emerged as a powerful tool for knock-in of DNA fragments via donor plasmid and homology-independent DNA repair mechanism; however, conventional integration includes unnecessary plasmid backbone and may result in the unfaithful expression of the modified endogenous genes. Here, we report an efficient and precise CRISPR/Cas9-mediated integration strategy using a donor plasmid that harbors 2 of the same cleavage sites that flank the cassette at both sides. After the delivery of donor plasmid, together with Cas9 mRNA and guide RNA, into cells or fertilized eggs, concurrent cleavages at both sides of the exogenous cassette and the desired chromosomal site result in precise targeted integration without plasmid backbone. We successfully used this approach to precisely integrate the EGFP reporter gene into the myh6 locus or the GAPDH locus in Xenopus tropicalis or human cells, respectively. Furthermore, we demonstrate that replacing conventional terminators with the endogenous 3UTR of target genes in the cassette greatly improves the expression of reporter gene after integration. Our efficient and precise method will be useful for a variety of targeted genome modifications, not only in X. tropicalis, but also in mammalian cells, and can be readily adapted to many other organisms.",
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Mao, CZ, Zheng, L, Zhou, YM, Wu, HY, Xia, JB, Liang, CQ, Guo, XF, Peng, WT, Zhao, H, Cai, WB, Kim, SK, Park, KS, Cai, DQ & Qi, XF 2018, 'CRISPR/Cas9-mediated efficient and precise targeted integration of donor DNA harboring double cleavage sites in Xenopus tropicalis', FASEB Journal, vol. 32, no. 12, pp. 6495-6509. https://doi.org/10.1096/fj.201800093

CRISPR/Cas9-mediated efficient and precise targeted integration of donor DNA harboring double cleavage sites in Xenopus tropicalis. / Mao, Cheng Zhou; Zheng, Li; Zhou, Yi Min; Wu, Hai Yan; Xia, Jing Bo; Liang, Chi Qian; Guo, Xiao Fang; Peng, Wen Tao; Zhao, Hui; Cai, Wei Bin; Kim, Soo Ki; Park, Kyu Sang; Cai, Dong Qing; Qi, Xu Feng.

In: FASEB Journal, Vol. 32, No. 12, 12.2018, p. 6495-6509.

Research output: Contribution to journalArticle

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T1 - CRISPR/Cas9-mediated efficient and precise targeted integration of donor DNA harboring double cleavage sites in Xenopus tropicalis

AU - Mao, Cheng Zhou

AU - Zheng, Li

AU - Zhou, Yi Min

AU - Wu, Hai Yan

AU - Xia, Jing Bo

AU - Liang, Chi Qian

AU - Guo, Xiao Fang

AU - Peng, Wen Tao

AU - Zhao, Hui

AU - Cai, Wei Bin

AU - Kim, Soo Ki

AU - Park, Kyu Sang

AU - Cai, Dong Qing

AU - Qi, Xu Feng

PY - 2018/12

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N2 - The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system has emerged as a powerful tool for knock-in of DNA fragments via donor plasmid and homology-independent DNA repair mechanism; however, conventional integration includes unnecessary plasmid backbone and may result in the unfaithful expression of the modified endogenous genes. Here, we report an efficient and precise CRISPR/Cas9-mediated integration strategy using a donor plasmid that harbors 2 of the same cleavage sites that flank the cassette at both sides. After the delivery of donor plasmid, together with Cas9 mRNA and guide RNA, into cells or fertilized eggs, concurrent cleavages at both sides of the exogenous cassette and the desired chromosomal site result in precise targeted integration without plasmid backbone. We successfully used this approach to precisely integrate the EGFP reporter gene into the myh6 locus or the GAPDH locus in Xenopus tropicalis or human cells, respectively. Furthermore, we demonstrate that replacing conventional terminators with the endogenous 3UTR of target genes in the cassette greatly improves the expression of reporter gene after integration. Our efficient and precise method will be useful for a variety of targeted genome modifications, not only in X. tropicalis, but also in mammalian cells, and can be readily adapted to many other organisms.

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