A pipeline for rapidly generating genetically engineered mouse models of pancreatic cancer using in vivo CRISPR-Cas9-mediated somatic recombination

Abstract

Genetically engineered mouse models (GEMMs) that recapitulate the major genetic drivers in pancreatic ductal adenocarcinoma (PDAC) have provided unprecedented insights into the pathogenesis of this lethal neoplasm. Nonetheless, generating an autochthonous model is an expensive, time consuming and labor intensive process, particularly when tissue specific expression or deletion of compound alleles are involved. In addition, many of the current PDAC GEMMs cause embryonic, pancreas-wide activation or loss of driver alleles, neither of which reflects the cognate human disease scenario. The advent of CRISPR/Cas9 based gene editing can potentially circumvent many of the aforementioned shortcomings of conventional breeding schema, but ensuring the efficiency of gene editing in vivo remains a challenge. Here we have developed a pipeline for generating PDAC GEMMs of complex genotypes with high efficiency using a single "workhorse" mouse strain expressing Cas9 in the adult pancreas under a p48 promoter. Using adeno-associated virus (AAV) mediated delivery of multiplexed guide RNAs (sgRNAs) to the adult murine pancreas of p48-Cre; LSL-Cas9 mice, we confirm our ability to express an oncogenic Kras G12D allele through homology-directed repair (HDR), in conjunction with CRISPRinduced disruption of cooperating alleles (Trp53, Lkb1 and Arid1A). The resulting GEMMs demonstrate a spectrum of precursor lesions (pancreatic intraepithelial neoplasia [PanIN] or Intraductal papillary mucinous neoplasm [IPMN] with eventual progression to PDAC. Next generation sequencing of the resulting murine PDAC confirms HDR of oncogenic Kras G12D allele at the endogenous locus, and insertion deletion ("indel") and frameshift mutations of targeted tumor suppressor alleles. By using a single "workhorse" mouse strain and optimal AAV serotype for in vivo gene editing with combination of driver alleles, we present a facile autochthonous platform for interrogation of the PDAC genome.

Paradoxical role of AT-rich interactive domain 1A in restraining pancreatic carcinogenesis

“…It is worth noting that in at least one prior study 8 , the "KAC" genotype rarely progressed to invasive cancers, with mice mostly developing IPMN precursors, unless additionally crossed to a mutant Trp53 background (providing one prototypal "escape" mechanism). Similarly, our recently published CRISPR-based mouse model of PDAC 53 showed Arid1a loss concurrent with oncogenic Kras mutation in adult acinar tissue only caused LG-PanINs, while emergence of well-differentiated PDAC in the same period required deletion of TRP53, irrespective of Arid1a loss. The distinction between the two scenarios -whether Arid1a loss cooperates with oncogenic Ras to induce PDAC formation (as proposed [5][6][7][8] ), or invasive cancers arise via an "escape" phenomenon in the setting of growth constrained precursors -goes beyond semantics, given the occurrence of ARID1A mutations across a multitude of epithelial pre-cancers 54 .…”

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

“…In the additional analysis for Kras G12D mutation, mice in all the groups exhibited low frequency of mutation. This SNP and indel frequency seemed to be lower than that recently reported in a study [17]. However, we collected whole pancreatic tissue for analyzing overall gene editing efficiency, but previous studies analyzed genomic DNA from neoplasm tissues, and this difference would probably have led to relatively low in vivo targeting efficiency.…”

“…Even mutation of each candidate genes enrolled in specific PanIN stage, we tried to develop simultaneous multiplex gene mutation for avoiding multiple surgery for AAV transduction. AAV-SpCas9 and AAV-CjCas9 developed PanIN, but there is no evidence of metastasis and overall progression is slower than previous in vivo pancreatic cancer modeling [17,29]. The reason for the slow PanIN development is still uncertain, but the transduction with lower AAV particles number than other studies and the low frequency of Kras G12D would cause this.…”

“…SpCas9 has simple PAM (5′-NGG-3′) and develops a relatively higher DSB frequency than other CRISPR orthologues that have been previously reported for in vivo cancer modeling utilizing SpCas9 orthologues [12,15,16,17,29]. In this study, we also applied SpCas9 for DSB formation for the selected targets.…”

“…Thus, in vivo somatic cell gene engineering with CRISPR has been receiving attention for generation of cancer in various organs including the lung, liver, brain, retina, and pancreas [11][12][13][14][15][16]. Even technical advances have been made in in vivo PDAC modeling, but a model could be developed based on pre-established Cas9 overexpression, Kras or p53 mice [15,17].…”