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

• Published in: iScience 2024-01, Vol.27 (1), p.108693-108693, Article 108693
• Main Authors: Bevacqua, Romina J., Zhao, Weichen, Merheb, Emilio, Kim, Seung Hyun, Marson, Alexander, Gloyn, Anna L., Kim, Seung K.
• Format: Article
• Language: English
• Subjects: biology experimental methods; cell biology; human genetics; Techniques in genetics
• ISSN: 2589-0042; 2589-0042
• DOI: 10.1016/j.isci.2023.108693

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. [Display omitted] •CRISPR/Cas9 RNP editing of coding and non-coding DNA in primary human islet cells•Identified MPHOSPH9 as an unsuspected regulator of β cell insulin secretion•CRISPR/Cas9 RNP allows multiplexed targeting of PCSK1 cis-regulatory elements•Identified cis-regulatory elements regulating glucose-dependent PCSK1 expression Techniques in genetics; Human Genetics; Cell biology; Biology experimental methods