Discovery of gene modifications that protect beta cells against autoimmunity by genome-wide CRISPR screening

Background

The CRISPR-Cas9 system has been developed into a phenotype screening strategy. Using lentiviral delivery of a large collection of gRNAs, thousands of genes can be targeted for Cas9-mediated disruption in mammalian cells. Growing evidence supports ER stress as a target to prevent the initiation of the autoimmune reaction, propagation of inflammation, and b cell death in type 1 diabetes.

Methods

We used a genome-wide library of more than 60,000 gRNAs to perform a loss-of-function screen in NIT-1 beta cells. We implanted mutant NIT-1 cells into NOD.scid mice in which we also transplanted splenocyte from diabetic NOD mice. We recovered surviving NIT-1 beta cells two months after implantation and extracted genomic DNA from explanted cells to measure gRNA enrichment by next-generation sequencing.

Results

We identified 13 gRNAs covering 12 genes in beta cells that survived autoimmunity in transplanted mice. Several of these gene mutations conferred resistance to ER stress-induced cell death, and one mutation abrogated both CD4⁺ and CD8⁺ T cell activation by NIT-1 cell stimulation in vitro. Significantly, two of the genes identified in this genome-wide screen have been associated with human diabetes by GWAS.

We have identified a tractable number of candidate genes whose disruption extended the survival of beta cells in mice with autoimmune diabetes. Two of these candidate genes are associated with human diabetes, suggesting that our screening strategy discovered relevant targets for the protection of beta cells in type 1 diabetes.