A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering
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 {beta} cells to secrete protective immunomodulatory factor interleukin-10.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC=\"FIGDIR/small/732354v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (30K):\norg.highwire.dtl.DTLVardef@48a34aorg.highwire.dtl.DTLVardef@d886d0org.highwire.dtl.DTLVardef@17acc61org.highwire.dtl.DTLVardef@fffe44_HPS_FORMAT_FIGEXP M_FIG TOC GRAPHIC C_FIG