Multiplexed CRISPR gene editing in primary human islet cells with Cas9 ribonucleoprotein.

Successful genome editing in primary human islets could reveal features of the genetic regulatory landscape underlying β cell function and diabetes risk. Here, we describe a CRISPR-based strategy to interrogate functions of predicted regulatory DNA elements using electroporation of a complex of Cas9 ribonucleoprotein (Cas9 RNP) and guide RNAs into primary human islet cells. We successfully targeted coding regions including the exon 1, and non-coding DNA linked to diabetes susceptibility. CRISPR-Cas9 RNP approaches revealed genetic targets of regulation by DNA elements containing candidate diabetes risk SNPs, including an enhancer of the gene. CRISPR-Cas9 RNP multiplexed targeting of two -regulatory elements linked to diabetes risk in , which encodes an endoprotease crucial for Insulin processing, also demonstrated efficient simultaneous editing of regulatory elements, resulting in impaired β cell regulation and Insulin secretion. Multiplex CRISPR-Cas9 RNP provides powerful approaches to investigate and elucidate human islet cell gene regulation in health and diabetes.