CRISPR-based genome editing in primary human pancreatic islet cells

Abstract

Gene targeting studies in primary human islets could advance our understanding of mechanisms driving diabetes pathogenesis. Here, we demonstrate successful genome editing in primary human islets using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). CRISPR-based targeting efficiently mutated protein-coding exons, resulting in acute loss of islet β-cell regulators, like the transcription factor PDX1 and the KATPchannel subunit KIR6.2, accompanied by impaired β-cell regulation and function. CRISPR targeting of non-coding DNA harboring type 2 diabetes (T2D) risk variants revealed changes inABCC8,SIX2andSIX3expression, and impaired β-cell function, thereby linking regulatory elements in these target genes to T2D genetic susceptibility. Advances here establish a paradigm for genetic studies in human islet cells, and reveal regulatory and genetic mechanisms linking non-coding variants to human diabetes risk.

Islet Biology During COVID-19: Progress and Perspectives

“…Representing a major advancement, the genome-editing technology CRISPR-Cas9 was used to generate the first genetically modified primary human islets (41). As a proof of concept, Bevacqua et al deleted pancreatic and duodenal homeobox 1 (PDX1), a transcription factor important for maintaining b-cell identity and function, impairing calcium-channel activity, reducing total insulin content and glucose-stimulated insulin secretion (41).…”

“…Representing a major advancement, the genome-editing technology CRISPR-Cas9 was used to generate the first genetically modified primary human islets (41). As a proof of concept, Bevacqua et al deleted pancreatic and duodenal homeobox 1 (PDX1), a transcription factor important for maintaining b-cell identity and function, impairing calcium-channel activity, reducing total insulin content and glucose-stimulated insulin secretion (41). CRISPR-Cas9 constructs delivered by lentivirus were also used to delete the KCNJ11 gene encoding K ATP channel subunit KIR6.2, or to introduce noncoding genetic variation at the ABCC8eKCNJ11 locus, both of which caused impaired K ATP channel activity in human islets (41,42).…”

“…As a proof of concept, Bevacqua et al deleted pancreatic and duodenal homeobox 1 (PDX1), a transcription factor important for maintaining b-cell identity and function, impairing calcium-channel activity, reducing total insulin content and glucose-stimulated insulin secretion (41). CRISPR-Cas9 constructs delivered by lentivirus were also used to delete the KCNJ11 gene encoding K ATP channel subunit KIR6.2, or to introduce noncoding genetic variation at the ABCC8eKCNJ11 locus, both of which caused impaired K ATP channel activity in human islets (41,42). Successful editing of the human islet genome via CRISPR-Cas9 provides proof of principle for generating a wide variety of human primary islet knock-in and knock-out models, which will help bridge the gap between in vitro models and human islet biology.…”

“…For the first time, researchers have used a Nobel Prizeewinning technique called CRISPR-Cas9emediated gene editing (see CRISPR-Cas9 explained for an easy-to-follow primer on how this technology works) to modify genes in pancreatic islets from human donors (41). This allows islet biologists to quickly modify specific genes in human islet cells to understand their importance.…”

“…This allows islet biologists to quickly modify specific genes in human islet cells to understand their importance. This technology offers the potential to allow scientists to improve human donorderived pancreatic islets, such as by repairing harmful mutations, enhancing function or improving survival (41). Although this represents a major advancement, we are again reminded of the continual need to revisit and discuss ethical and socioeconomic concerns that underlie gene therapy.…”