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

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) 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.

Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo

“…Delivery of most of these compact Cas9s within single-vector AAVs, in which both the Cas9 and its sgRNA are packaged into the same virion, has been validated in vivo [22][23][24][27][28][29] . AAV vectors encoding SauCas9 along with two sgRNAs 30,31 , as well as CjeCas9 with three sgRNAs 32 , have also been reported, enabling the introduction of multiplexed edits or segmental deletions. A few of the smaller Cas9s are also less prone to off-target cellular editing than wild-type SpyCas9 23,24,27,33,34 , even with guides that support efficient on-target editing, likely because of reduced enzymatic efficiencies that disproportionately dampen activity at near-cognate sites 35 .…”

MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression in vivo

“…More importantly, delivered via AAV, Cj Cas9 can induce indels in mouse muscle cells to treat Duchenne muscular dystrophy (DMD) and in retinal pigment epithelium (RPE) cells to suppress pathological choroidal neovascularization ( 19 , 22 , 23 ). Additionally, pancreatic cancer modeling can be achieved by in vivo multiplex gene editing with Cj Cas9 ( 24 ). Therefore, Cj Cas9 has been well adopted for efficient genome editing in vivo .…”

An Exploration of Oral-Gut Pathogens Mediating Immune Escape of Pancreatic Cancer via miR-21/PTEN Axis

“…Campylobacter jejuni is a bacterial enteric pathogen often causing diarrhea and enterocolitis, and its Cas9 protein contributes to the carcinogenesis of pancreatic cancer (Chang et al, 2020 ). Clostridium difficile is a bacterium that causes mild to severe diarrhea and its infection is mostly found in patients with pancreatic ductal adenocarcinoma (PDAC) (Sumiyoshi et al, 2022 ).…”