Functional Genomics in Pancreatic β Cells: Recent Advances in Gene Deletion and Genome Editing Technologies for Diabetes Research

Abstract

The inheritance of variants that lead to coding changes in, or the mis-expression of, genes critical to pancreatic beta cell function can lead to alterations in insulin secretion and increase the risk of both type 1 and type 2 diabetes. Recently developed clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) gene editing tools provide a powerful means of understanding the impact of identified variants on cell function, growth, and survival and might ultimately provide a means, most likely after the transplantation of genetically "corrected" cells, of treating the disease. Here, we review some of the disease-associated genes and variants whose roles have been probed up to now. Next, we survey recent exciting developments in CRISPR/Cas9 technology and their possible exploitation for β cell functional genomics. Finally, we will provide a perspective as to how CRISPR/Cas9 technology may find clinical application in patients with diabetes.

Keywords: beta cell; genome editing; genome-wide association studies; maturity onset of diabetes of the young; mouse models; stem cells.

Figure 1

A versatile genome-editing toolbox. Following the original demonstration of genome editing, applications of ZFN, TALEN, and CRISPR for genome editing, regulation, monitoring, and beyond were subsequently developed (49). Conceptually, there are three major application tracks: 1. genome editing, including gene knockout, knockin, and indel formation initiated with a double strand DNA break made by a nuclease; 2. gene regulation and delivery of various functional moieties (e.g., transcription factors) to unique sites in DNA using catalytically inactivated derivatives of the same nucleases; 3. targeting single strand RNA for inactivation, editing, modification, or localization.

Figure 2

Generation, differentiation, and transplantation of iPSC cells into diabetic patients. A biopsy (skin fibroblasts, cord blood, or peripheral blood cells) is obtained from diabetes patient and cultured in the laboratory. Cultured cells are reprogrammed into iPSC cells using Yamanaka factors (271). To correct genetic mutation(s) or convert risk variant(s) into protective variant(s), iPSC can be edited at the genome level using one of the genome-editing tools. Engineered iPSC cells are then differentiated into β cell-like cells through a stepwise differentiation protocol by exposing the cells to specific growth factors/cytokines and signalling proteins. Alternatively, hESCs derived from healthy individuals can be cultured in the laboratory and driven to differentiate towards mature β cells. Those in vitro generated β cell-like cells which are free of mutation(s) can then be transplanted into patients to reverse diabetes. In addition, hESCs can be genome-edited in reverse to create disease-related mutation(s) and then subjected to directed β cell differentiation. Such mutation-bearing β cell-like cells can be used for the molecular mechanistic studies of disease as well as drug screening to identify therapeutic reagents.