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Engineering designer beta cells with a CRISPR-Cas9 conjugation platform

Author

Listed:
  • Donghyun Lim

    (Broad Institute of MIT and Harvard
    Harvard Medical School
    Brigham and Women’s Hospital)

  • Vedagopuram Sreekanth

    (Broad Institute of MIT and Harvard
    Harvard Medical School
    Brigham and Women’s Hospital)

  • Kurt J. Cox

    (Broad Institute of MIT and Harvard
    Harvard Medical School
    Brigham and Women’s Hospital)

  • Benjamin K. Law

    (Broad Institute of MIT and Harvard
    Harvard Medical School
    Brigham and Women’s Hospital)

  • Bridget K. Wagner

    (Broad Institute of MIT and Harvard)

  • Jeffrey M. Karp

    (Center for Regenerative Therapeutics, Brigham and Women’s Hospital, Harvard Medical School
    MIT
    Proteomics Platform, Broad Institute of MIT and Harvard
    Harvard University)

  • Amit Choudhary

    (Broad Institute of MIT and Harvard
    Harvard Medical School
    Brigham and Women’s Hospital)

Abstract

Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, the genetic modifications are limited to natural polypeptide chains at the Cas9 termini, which excludes a diverse array of molecules useful for gene editing. Here, we report chemical modifications that allow site-specific and multiple-site conjugation of a wide assortment of molecules on both the termini and internal sites of Cas9, creating a platform for endowing Cas9 with diverse functions. Using this platform, Cas9 can be modified to more precisely incorporate exogenously supplied single-stranded oligonucleotide donor (ssODN) at the DNA break site. We demonstrate that the multiple-site conjugation of ssODN to Cas9 significantly increases the efficiency of precision genome editing, and such a platform is compatible with ssODNs of diverse lengths. By leveraging the conjugation platform, we successfully engineer INS-1E, a β-cell line, to repurpose the insulin secretion machinery, which enables the glucose-dependent secretion of protective immunomodulatory factor interleukin-10.

Suggested Citation

  • Donghyun Lim & Vedagopuram Sreekanth & Kurt J. Cox & Benjamin K. Law & Bridget K. Wagner & Jeffrey M. Karp & Amit Choudhary, 2020. "Engineering designer beta cells with a CRISPR-Cas9 conjugation platform," Nature Communications, Nature, vol. 11(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-17725-0
    DOI: 10.1038/s41467-020-17725-0
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    Cited by:

    1. Aron Ferenczi & Yen Peng Chew & Erika Kroll & Charlotte Koppenfels & Andrew Hudson & Attila Molnar, 2021. "Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii," Nature Communications, Nature, vol. 12(1), pages 1-12, December.

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