Engineering designer beta cells with a CRISPR-Cas9 conjugation platform
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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Aug 13, 2020
- Source ID
- 10.1038/s41467-020-17725-0
Entities
People
- Amit Choudhary
- Benjamin K. Law
- Bridget K Wagner
- Donghyun Lim
- Jeffrey Karp
- Kurt J Cox
- Vedagopuram Sreekanth
Organizations
- United States Department of Defense