J Vet Sci.  2020 Mar;21(2):e26. 10.4142/jvs.2020.21.e26.

in vivo multiplex gene targeting with Streptococcus pyogens and Campylobacter jejuni Cas9 for pancreatic cancer modeling in wildtype animal

Affiliations
  • 1Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang 25354, Korea
  • 2Department of Pharmacology, Yonsei University College of Medicine, Seoul 03722, Korea
  • 3Toolgen Inc., Seoul 08594, Korea
  • 4Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University, Seoul 05029, Korea
  • 5Designed Animal and Transplantation Research Institute, Greenbio Research and Technology, Seoul National University, Pyeongchang 25354, Korea

Abstract

Pancreatic ductal adenocarcinoma is a lethal cancer type that is associated with multiple gene mutations in somatic cells. Genetically engineered mouse is hardly applicable for developing a pancreatic cancer model, and the xenograft model poses a limitation in the reflection of early stage pancreatic cancer. Thus, in vivo somatic cell gene engineering with clustered regularly interspaced short palindromic repeats is drawing increasing attention for generating an animal model of pancreatic cancer. In this study, we selectedKras, Trp53, Ink4a, Smad4, and Brca2 as target genes, and applied Campylobacter jejuni Cas9 (CjCas9) andStreptococcus pyogens Cas9 (SpCas9) for developing pancreatic cancer using adeno associated virus (AAV) transduction. After confirming multifocal and diffuse transduction of AAV2, we generated SpCas9 overexpression mice, which exhibited high double-strand DNA breakage (DSB) in target genes and pancreatic intraepithelial neoplasia (PanIN) lesions with two AAV transductions; however, wild-type (WT) mice with three AAV transductions did not develop PanIN. Furthermore, small-sized Cjcas9 was applied to WT mice with two AAV system, which, in addition, developed high extensive DSB and PanIN lesions. Histological changes and expression of cancer markers such as Ki67, cytokeratin, Mucin5a, alpha smooth muscle actin in duct and islet cells were observed. In addition, the study revealed several findings such as 1) multiple DSB potential of AAV-CjCas9, 2) peri-ductal lymphocyte infiltration, 3) multi-focal cancer marker expression, and 4) requirement of > 12 months for initiation of PanIN in AAV mediated targeting. In this study, we present a useful tool for in vivo cancer modeling that would be applicable for other disease models as well.

Keyword

Adeno-associated virus; CRISPR; gene editing; Pancreatic cancer
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