Multiplexed CRISPR gene editing in primary human islet cells with Cas9 ribonucleoprotein.
iScience
; 27(1): 108693, 2024 Jan 19.
Article
en En
| MEDLINE
| ID: mdl-38205242
ABSTRACT
Successful genome editing in primary human islets could reveal features of the genetic regulatory landscape underlying ß 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 ß 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.
Texto completo:
1
Bases de datos:
MEDLINE
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
IScience
Año:
2024
Tipo del documento:
Article
País de afiliación:
Estados Unidos