Engineering designer beta cells with a CRISPR-Cas9 conjugation platform

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 th...

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Bibliographic Details
Published in:Nature communications 2020-08, Vol.11 (1), p.4043-4043, Article 4043
Main Authors: Lim, Donghyun, Sreekanth, Vedagopuram, Cox, Kurt J., Law, Benjamin K., Wagner, Bridget K., Karp, Jeffrey M., Choudhary, Amit
Format: Article
Language:English
Subjects:
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Beta cells
Chains
Conjugation
CRISPR
Deoxyribonucleic acid
DNA
DNA damage
Engineers
Genetic modification
Genome editing
Genomes
Humanities and Social Sciences
Immunomodulation
Insulin
Insulin secretion
Interleukin 10
multidisciplinary
Oligonucleotides
Polypeptides
Science
Science (multidisciplinary)
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Summary: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. Cas9 fusions partners are often limited to natural polypeptide chains at the Cas9 termni. Here the authors present a platform for site-specific and multiple-site conjugation to both termini and internal sites of Cas9, and they apply this platform to efficiently engineer insulin-producing β cells.
ISSN:2041-1723
2041-1723
DOI:10.1038/s41467-020-17725-0