Summary 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 PDX1 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 in vivo enhancer of the MPHOSPH9 gene. CRISPR/Cas9 RNP multiplexed targeting of two cis -regulatory elements linked to diabetes risk in PCSK1 , which encodes an endoprotease crucial for insulin processing, also demonstrated efficient simultaneous editing of PCSK1 regulatory elements, resulting in impaired β cell PCSK1 regulation and insulin secretion. Multiplex CRISPR/Cas9 RNP provides powerful approaches to investigate and elucidate human islet cell gene regulation in health and diabetes.

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