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- Title
Engineering designer beta cells with a CRISPR-Cas9 conjugation platform.
- Authors
Lim, Donghyun; Sreekanth, Vedagopuram; Cox, Kurt J.; Law, Benjamin K.; Wagner, Bridget K.; Karp, Jeffrey M.; Choudhary, Amit
- 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. 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.
- Subjects
PANCREATIC beta cells; PROTEIN domains; GENOME editing; GLUCOSE analysis; INTERLEUKIN-10; DESIGNERS
- Publication
Nature Communications, 2020, Vol 11, Issue 1, p1
- ISSN
2041-1723
- Publication type
Article
- DOI
10.1038/s41467-020-17725-0