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

Romina J Bevacqua; Weichen Zhao; Emilio Merheb; Seung Hyun Kim; Alexander Marson; Anna L Gloyn; Seung K Kim

doi:10.1101/2023.09.16.558090

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

bioRxiv [Preprint]. 2023 Sep 17:2023.09.16.558090. doi: 10.1101/2023.09.16.558090.

Authors

Romina J Bevacqua^{1

2

3}, Weichen Zhao¹, Emilio Merheb², Seung Hyun Kim¹, Alexander Marson^{4

5

6

7}, Anna L Gloyn^{8

9}, Seung K Kim^{1

9

10

11}

Affiliations

¹ Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA, 94305, USA.
² Diabetes, Obesity and Metabolism Institute (DOMI), Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
³ Mount Sinai Regenerative Biology and Stem Cell Institute, New York, NY, United States.
⁴ Gladstone-UCSF Institute of Genomic Immunology and Northern California JDRF Center of Excellence, University of California at San Francisco, CA, 94158, USA.
⁵ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
⁶ Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA.
⁷ Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA.
⁸ Department of Pediatrics (Endocrinology) and of Genetics, Stanford University School of Medicine, Stanford, CA, 94305, USA.
⁹ Departments of Medicine and of Pediatrics, Stanford University School of Medicine, Stanford, CA, 94305, USA.
¹⁰ Stanford Diabetes Research Center, Stanford University School of Medicine, Stanford, CA, 94305, USA.
¹¹ Northern California JDRF Center of Excellence, Stanford University School of Medicine, Stanford, CA, 94305, USA.

Abstract

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.

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