In vivo multiplex gene targeting with Streptococcus pyogens and Campylobacter jejuni Cas9 for pancreatic cancer modeling in wild-type animal

Yoo Jin Chang; Jihyeon Bae; Yang Zhao; Geonseong Lee; Jeongpil Han; Yoon Hoo Lee; Ok Jae Koo; Sunmin Seo; Yang-Kyu Choi; Su Cheong Yeom

doi:10.4142/jvs.2020.21.e26

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Chang, Bae, Zhao, Lee, Han, Lee, Koo, Seo, Choi, and Yeom: In vivo multiplex gene targeting with Streptococcus pyogens and Campylobacter jejuni Cas9 for pancreatic cancer modeling in wild-type animal

Original Article

J Vet Sci 2020;21(2):e26.

Published online: 4 March 2020

DOI: https://doi.org/10.4142/jvs.2020.21.e26

In vivo multiplex gene targeting with Streptococcus pyogens and Campylobacter jejuni Cas9 for pancreatic cancer modeling in wild-type animal

Yoo Jin Chang^1,², Jihyeon Bae¹, Yang Zhao¹, Geonseong Lee¹, Jeongpil Han¹, Yoon Hoo Lee¹, Ok Jae Koo³, Sunmin Seo⁴, Yang-Kyu Choi⁴, Su Cheong Yeom^1,^5,^*

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

^*Corresponding author: Su Cheong Yeom Graduate School of International Agricultural Technology, Seoul National University, 1447 Pyeongchang-daero, Daewha-myeon, Pyeongchang 25354, Korea. E-mail: scyeom@snu.ac.kr

Conflict of Interest The authors declare no conflicts of interest.

Received 23 July 2019 Revised 4 December 2019 Accepted 6 December 2019

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 selected Kras, Trp53, Ink4a, Smad4, and Brca2 as target genes, and applied Campylobacter jejuni Cas9 (CjCas9) and Streptococcus 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) periductal 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.

Keywords: Adeno-associated virus, CRISPR, gene editing, Pancreatic cancer

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Fig. 1.

Brief schematic of in vivo gene targeting with CRISPR/Cas9 (A) Vector maps of AAV-SpCas9, AAV-3 sgRNA, and AAV- CjCas9-all-in-one used in this study. Five different rAAV vectors were prepared by synthesis and cloning. Blue box: ITR sequence from AAV2, arrow box: promoter, brown box: sgRNA sequence. (B) SpCas9 overexpression mouse generation with PiggyBac transposon system. pCAG-SpCas9-RFP-pA insert was incorporated into host genome by transposase. Gene integration confirmed by RFP expression under stereoscope microscope. (C) Experimental group of in vivo gene targeting for pancreatic cancer development. WT C57BL/6 mice were injected with three AAVs; AAV2-SpCas9, AAV2-3 sgRNA (Kras-Ink4a1-In4a2-HA) and AAV2-3 sgRNA (Trp53-Smad4-Brca2) into common bile duct in group 1. Instead of AAV2-SpCas9 injection, SpCas9 overexpression mice were applied in group 2. WT FVB mice and all-in-one type two AAVs (AAV2-CjCas9-sgRNA for Kras, Ink4a-HA and AAV2-CjCas9-sgRNA for Trp53, Smad4, Brca2) were injected in group 3. Detailed information of each target is provided in Supplementary Fig. 1 and Supplementary Table 1. AAV, adeno associated virus; SpCas9, Streptococcus pyogens Cas9; CjCas9, Campylobacter jejuni Cas9; sgRNA, single guide RNA; CRISPR, clustered regularly interspaced short palindromic repeats; rAAV, recombinant adeno associated virus; WT, wild-type.

Fig. 2.

Evaluation of DSB efficiency and AAV2 tropism in pancreas (A) and (B). Cas9 messenger RNA and 3 single guide RNAs plasmid were co-injected into 1 cell stage embryos and DSB was evaluated by hetero-duplex formation and SDS-PAGE gel. Red letters indicate DSB and black arrow indicates the target size of each polymerase chain reaction reaction. DSB efficiency presented as number of embryos with DSB/number of total embryos used (%). (C) After 3 weeks of AAV2-eGFP injection into pancreas, GFP expression analyzed with stereoscopic microscope. White guideline distinguishes each of the organs and white arrows indicates spots for GFP expression. Statistical analysis was performed using Student's t-test. SpCas9, Streptococcus pyogens Cas9; CjCas9, Campylobacter jejuni Cas9; DSB, double-strand DNA breakage; SDS-PAGE, sodium dodecyl sulfate– polyacrylamide gel electrophoresis; AAV, adeno associated virus; eGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; WT, wild-type.

Fig. 3.

DSB frequency analysis and macroscopic examination (A) Indel frequency of target genes was analyzed with Sanger sequencing and algorithm of Synthego ICE tool. Each dot indicates DSB rate from each mice. Statistical analysis was performed with Student's t-test, and p < 0.05 indicated significantly different. (B) Appearance and microscopic analysis on pancreas. Red arrow: abnormal lesion, yellow arrow: islet hyperplasia, black arrow: periductal lymphocyte infiltration and mesentery lymph node, white bar, 200 µm. DSB, double-strand DNA breakage.

Fig. 4.

Histological and IHC analysis for evaluation of pancreatic cancer development (A) and (B) H&E stain and IHC for pan-cytokeratin, α SMA, Mucin5a, and Ki67 was conducted with formalin fixed pancreatic tissues. Black arrow: PanIN lesion or cancer marker expression. Black scale bar: 50 µm (C) IHC for α SMA marker. Yellow scale bar, 100 µm. IHC, immunohistochemistry; α SMA, alpha smooth muscle actin; H&E, hematoxylin and eosin; PanIN, pancreatic intraepithelial neoplasia.

Table 1.

Summary of in vivo gene targeting for developing pancreatic cancer

Group	Strain	Component	6 (mo)	12 (mo)
Group 1	B6	AAV-SpCas9	–	Lymphoid cell infiltration
		AAV-Ink4a1-Ink4a2-Kras-Kras HA		Cancer marker expression (α SMA)
		AAV-Tp53-Smad4-Brca2
Group 2	B6.PB-SpCas9	AAV-Ink4a1-Ink4a2-Kras-Kras HA	Islet hyperplasia	Mesentery lymph node penetration, PanIN, cancer marker
		AAV-Tp53-Smad4-Brca2		expression (Ki67, cytokeratin, Mucin5a, α SMA)
Group 3	FVB	AAV-Ink4a-Kras1-Kras2-Kras HA	Lymphoid cell infiltrati	ion Mesentery lymph node penetration, PanIN, cancer marker
		AAV-Tp53-Smad4-Brca2		expression (Ki67, cytokeratin, α SMA)

AAV, adeno associated virus; SpCas9, Streptococcus pyogens Cas9; α SMA, alpha smooth muscle actin; PanIN, pancreatic intraepithelial neoplasia.

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