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.

CRISPR/Cas based gene editing: marking a new era in medical science

“…Another example of therapeutic cells is engineered IPSCs which has then been differentiated to pancreatic beta cells for the treatment of diabetes. IPSCs in theory can be differentiated into any cell type and can be used for cell based therapy for human diseases [ 90 , 91 ].…”

Genome engineering and disease modeling via programmable nucleases for insulin gene therapy; promises of CRISPR/Cas9 technology

“…As isolated human islets are rare and valuable materials for diabetes research, hPSCs represent a good alternative to pancreatic islet cell donors. iPSCs can be established from adult somatic cells via direct reprogramming and differentiated into beta cell-like insulin-producing cells[ 88 ]. Although full maturation of these cells into IPCs that secrete insulin in response to changes in blood glucose concentration cannot be achieved easily, this approach can be used to model developmental defects of genetic diseases like diabetes[ 68 ].…”

CRISPR-based strategies for targeted transgene knock-in and gene correction

“…The use of this Cas9-DN1S fusion protein can circumvent the issue associated with the unwanted effects of global NHEJ inhibition. Another strategy to improve HDR efficiency is to covalently link the DNA repair templates to CRISPR/Cas9 complexes 87 – 89 . For example, to shuttle the HDR template to the nucleus, truncated Cas9 target sequences (16 bp) are added to the ends of the template for interacting with Cas9 RNPs ( Figure 1G ) 90 .…”

“…Because Cas9 contains nuclear localization sequences (NLSs), Cas9 RNPs along with the associated HDR template can be transported together into the cell nucleus. To further enhance HDR efficiency, multiple copies of DNA repair templates are conjugated to multiple sites on a Cas9 via the conjugation adaptor 89 . Each conjugation adaptor on Cas9 bears the complementary sequence to the DNA repair template and thus multiple conjugation adaptors enable multivalent display of DNA repair template on Cas9.…”