Engineering designer beta cells with a CRISPR-Cas9 conjugation platform

• Published in: Nature communications Vol. 11; no. 1; p. 4043
• Main Authors: Lim, Donghyun, Sreekanth, Vedagopuram, Cox, Kurt J., Law, Benjamin K., Wagner, Bridget K., Karp, Jeffrey M., Choudhary, Amit
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group 13.08.2020; Nature Publishing Group UK; Nature Portfolio
• Subjects: Beta cells; Chains; Conjugation; CRISPR; Deoxyribonucleic acid; DNA; DNA damage; Engineers; Genetic modification; Genome editing; Genomes; Immunomodulation; Insulin; Insulin secretion; Interleukin 10; Oligonucleotides; Polypeptides
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-020-17725-0

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.