A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

Abstract

Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, these fusions are polypeptidic, limited to the Cas9 termini and lack multivalent display, and exclude diverse array of molecules. Here, we report a platform for the site-specific and multivalent display of a wide assortment of molecules on both the termini and internal sites on Cas9. Using this platform, we endow Cas9 with the functionality to effect precision genome edits, which involves efficient incorporation of exogenously supplied single-stranded oligonucleotide donor (ssODN) at the break site. We demonstrate that the multivalent display of ssODN on Cas9 significantly increased precision genome edits over those of Cas9 bearing one or no ssODN, and such display platform is compatible with large oligonucleotides and rapid screening of ssODNs. By hijacking the insulin secretion machinery and leveraging the ssODN display platform, we successfully engineer pancreatic β cells to secrete protective immunomodulatory factor interleukin-10. TOC GRAPHIC MAINCRISPR-Cas9 is a DNA endonuclease that can be targeted to a genomic site using a guide RNA (gRNA) bearing sequence complementarity to the target site. 1 Genetic fusion of Cas9 with effector domains (e.g., a transcription activator) has yielded transformative technologies; 2,3 however, this approach is limited to fusions that are generally linear, polypeptidic, and located on the termini of Cas9. A conjugation platform that allows the creation of fusions that are non-polypeptidic (e.g., nucleic acids, small molecules, polyethylene glycol [PEG] chains), unnatural peptides/proteins, internally located on Cas9, and branched with a multivalent display, would provide a greater diversity of technologies and applications. For example, precise sequence alteration at the Cas9 cleavage site requires the efficient incorporation of exogenously supplied single-stranded oligonucleotide donor DNA (ssODN) 4 via the homology-directed repair (HDR) pathway. 5-7 However, most cells instead employ the nonhomologous end-joining (NHEJ) repair, which results in unpredictable insertions and deletions of bases at the cleavage site, some of which are large enough to have pathogenic consequences. 3,8,9 Displaying ssODNs on Cas9 can increase their local concentration around the DNA strand break site to allow enhanced incorporation of the desired sequence. In another application, appending PEG chains to Cas9 may reduce the immunogenicity of this bacterial protein. 10 Multivalent display of DNA repair pathway inhibitors (e.g., NHEJ or p53 pathway inhibitors) or cell-specific ligands could also enhance precision and efficacious genome editing. 11,12 An ideal conjugation platform for Cas9 should have several characteristics. First, the platform should be compatible with a diverse set of cargoes (e.g., small molecules, nucleic acids, nanoparticles, antibodies, PEG chains) and allow their multivalent display. Second, the platform should be robust and implementable by nonspecialists, given the diverse u...

Secreted cytokines provide local immune tolerance for human stem cell-derived islets

“…This finding has several important implications. First, since b cells are professional secretory cells with a significant translatory demand, it demonstrates that SC-islet cells can be co-opted to secrete other proteins while maintaining their designed function (Lim et al, 2020).…”