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Biallelic and gene-wide genomic substitution for endogenous intron and retroelement mutagenesis in human cells

Author

Listed:
  • Tomoyuki Ohno

    (Tokyo Institute of Technology
    Logomix, Inc.)

  • Taichi Akase

    (Tokyo Institute of Technology)

  • Shunya Kono

    (Tokyo Institute of Technology)

  • Hikaru Kurasawa

    (Tokyo Institute of Technology
    Kanagawa Institute of Industrial Science and Technology)

  • Takuto Takashima

    (Tokyo Institute of Technology)

  • Shinya Kaneko

    (Tokyo Institute of Technology)

  • Yasunori Aizawa

    (Tokyo Institute of Technology
    Logomix, Inc.
    Kanagawa Institute of Industrial Science and Technology)

Abstract

Functional annotation of the vast noncoding landscape of the diploid human genome still remains a major challenge of genomic research. An efficient, scarless, biallelic, and gene-wide mutagenesis approach is needed for direct investigation of the functional significance of endogenous long introns in gene regulation. Here we establish a genome substitution platform, the Universal Knock-in System or UKiS, that meets these requirements. For proof of concept, we first used UKiS on the longest intron of TP53 in the pseudo-diploid cell line HCT116. Complete deletion of the intron, its substitution with mouse and zebrafish syntenic introns, and specific removal of retrotransposon-derived elements (retroelements) were all efficiently and accurately achieved in both alleles, revealing a suppressive role of intronic Alu elements in TP53 expression. We also used UKiS for TP53 intron deletion in human induced pluripotent stem cells without losing their stemness. Furthermore, UKiS enabled biallelic removal of all introns from three human gene loci of ~100 kb and longer to demonstrate that intron requirements for transcriptional activities vary among genes. UKiS is a standard platform with which to pursue the design of noncoding regions for genome writing in human cells.

Suggested Citation

  • Tomoyuki Ohno & Taichi Akase & Shunya Kono & Hikaru Kurasawa & Takuto Takashima & Shinya Kaneko & Yasunori Aizawa, 2022. "Biallelic and gene-wide genomic substitution for endogenous intron and retroelement mutagenesis in human cells," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
  • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-31982-1
    DOI: 10.1038/s41467-022-31982-1
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    References listed on IDEAS

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    1. Matthew C. Canver & Elenoe C. Smith & Falak Sher & Luca Pinello & Neville E. Sanjana & Ophir Shalem & Diane D. Chen & Patrick G. Schupp & Divya S. Vinjamur & Sara P. Garcia & Sidinh Luc & Ryo Kurita &, 2015. "BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis," Nature, Nature, vol. 527(7577), pages 192-197, November.
    2. Julie Parenteau & Laurine Maignon & Mélodie Berthoumieux & Mathieu Catala & Vanessa Gagnon & Sherif Abou Elela, 2019. "Introns are mediators of cell response to starvation," Nature, Nature, vol. 565(7741), pages 612-617, January.
    3. Gill Bejerano & Craig B. Lowe & Nadav Ahituv & Bryan King & Adam Siepel & Sofie R. Salama & Edward M. Rubin & W. James Kent & David Haussler, 2006. "A distal enhancer and an ultraconserved exon are derived from a novel retroposon," Nature, Nature, vol. 441(7089), pages 87-90, May.
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