Generation of focal mutations and large genomic deletions in the pancreas using inducible in vivo genome editing

Abstract

Beyond the nearly uniform presence of KRAS mutations, pancreatic cancer is increasingly recognized as a heterogeneous disease. Preclinical in vivo model systems exist, but with the advent of precision oncology, murine models with enhanced genetic flexibility are needed to functionally annotate genetic alterations found in the human malignancy. Here, we describe the generation of focal gene disruptions and large chromosomal deletions via inducible and pancreas-specific expression of Cas9 in adult mice. Experimental mice are derived on demand directly from genetically engineered embryonic stem cells, without the need for further intercrossing. To provide initial validation of our approach, we show that disruption of the E3 ubiquitin ligase Rnf43 accelerates KrasG12D-dependent tumourigenesis. Moreover, we demonstrate that this system can be used to rapidly interrogate the impact of complex cancer-associated alleles through the generation of a previously unstudied 1.2 megabase deletion surrounding the CDKN2A and CDKN2B tumour suppressors. Thus, our approach is capable of reproducibly generating biallelic and precise loss of large chromosomal fragments that, in conjunction with mutant Kras, leads to development of pancreatic ductal adenocarcinoma with full penetrance.

MACHETE identifies interferon-encompassing chromosome 9p21.3 deletions as mediators of immune evasion and metastasis

“…CNA research has commonly focused on known drivers within the affected regions, yet co-gained or co-deleted genes-once considered 'passenger' events-can contribute to tumorigenesis 1,8,9 . These observations imply that CNAs produce complex phenotypes that cannot be recapitulated by manipulating single genes [10][11][12][13] ; however, modeling CNAs remains a major challenge that has impeded their functional assessment 11,12,[14][15][16] .…”

Chromosome 9p21.3 Coordinates Cell Intrinsic and Extrinsic Tumor Suppression

“…While much of the research on CNAs has focused on known drivers within the affected regions, emerging evidence indicates that co-gained or co-deleted genes -once considered "passenger" events -can also contribute to tumorigenesis 1,9,10 . While these observations imply CNAs produce complex phenotypes that cannot be recapitulated by manipulating a single gene [11][12][13][14][15][16][17] , the experimental modeling of these lesions remains a major challenge that has impeded the functional assessment of CNA biology 12,13,[18][19][20][21] .…”

WNT Ligand Dependencies in Pancreatic Cancer

“…Comprehensive sequencing and functional analysis suggest most RNF43 non-sense and frameshift mutations and missense mutations in its RING domain and N-terminal region increase WNT activity and predict in vivo response to upstream WNT pathway inhibition ( Yu et al, 2020 ). Large genomic deletion of Rnf43 by inducible CRISPR in KC mice does not lead to cystic neoplasia but does accelerate PDAC progression, implying WNT signaling also facilitates malignant progression of Kras- initiated PanIN ( Mishra et al, 2020 ).…”