Mutational analyses of novel rat models with targeted modifications in inflammatory bowel disease susceptibility genes.

Mutations and single base pair polymorphisms in various genes have been associated with increased susceptibility to inflammatory bowel disease (IBD). We have created a series of rat strains carrying targeted genetic alterations within three IBD susceptibility genes: Nod2, Atg16l1, and Il23r, using CRISPR/Cas9 genome editing technology. Knock-out alleles and alleles with known human susceptibility polymorphisms were generated on three different genetic backgrounds: Fischer, Lewis and Sprague Dawley. The availability of these rat models will contribute to our understanding of the basic biological roles of these three genes as well as provide new potential IBD animal models.

The Atg16l1 gene: characterization of wild type, knock-in, and knock-out phenotypes in rats.

ATG16L1 is a ubiquitous autophagy gene responsible, in part, for formation of the double-membrane bound autophagosome that delivers unwanted cellular debris and intracellular pathogens to the lysosome for degradation. A single, nonsynonymous adenine to guanine polymorphism resulting in a threonine to alanine amino acid substitution (T300A) directly preceded by a caspase cleavage site (DxxD) causes an increased susceptibility to Crohn's disease (CD) in humans. The mechanism behind this increased susceptibility is still being elucidated, however, the amino acid change caused by this point mutation results in increased ATG16L1 protein sensitivity to caspase 3-mediated cleavage. To generate novel rat strains carrying genetic alterations in the rat Atg16l1 gene, we first characterized the wild-type rat gene. We identified four alternative splice variants with tissue-specific expression. Using CRISPR-Cas9 genome editing technology, we developed a knock-in rat model for the human ATG16L1 T300A CD risk polymorphism, as well as a knock-out rat model to evaluate the role of Atg16l1 in autophagy as well as its potential effect on CD susceptibility. These are the first reported rat strains with alterations of the Atg16l1 gene. Consistent with studies of the effects of human ATG16L1 polymorphisms, models exhibit morphological abnormalities in both Paneth and goblet cells, but do not develop spontaneous intestinal permeability or inflammatory bowel disease. Analysis of the gut microbiota does not show inherent differences in bacterial composition between wild-type and genetically modified animals. These Atg16l1 strains are valuable new animal models for the study of both autophagy and CD susceptibility.

A novel conditional ZsGreen-expressing transgenic reporter rat strain for validating Cre recombinase expression.

The Cre/loxP recombination system has revolutionized the ability to genetically manipulate animal genomes in order to conditionally control gene expression. With recent advances in genome editing, barriers to manipulating the rat genome have been overcome and it is now possible to generate new rat strains (Cre drivers) in which Cre recombinase expression is carefully controlled temporally and/or spatially. However, the ability to evaluate and characterize these Cre driver strains is limited by the availability of reliable reporter rat strains. Here, we describe the generation and characterization of a new transgenic rat strain in which conditional expression of the ZsGreen fluorescent protein gene requires the presence of exogenous Cre recombinase. Breeding Cre-expressing rat strains to this stable ZsGreen reporter strain provides an ideal method for validating new rat Cre driver lines and will greatly accelerate the characterization pipeline.

Differential susceptibility to colorectal cancer due to naturally occurring gut microbiota.

Recent studies investigating the human microbiome have identified particular bacterial species that correlate with the presence of colorectal cancer. To evaluate the role of qualitatively different but naturally occurring gut microbiota and the relationship with colorectal cancer development, genetically identical embryos from the Polyposis in Rat Colon (Pirc) rat model of colorectal cancer were transferred into recipients of three different genetic backgrounds (F344/NHsd, LEW/SsNHsd, and Crl:SD). Tumor development in the pups was tracked longitudinally via colonoscopy, and end-stage tumor burden was determined. To confirm vertical transmission and identify associations between the gut microbiota and disease phenotype, the fecal microbiota was characterized in recipient dams 24 hours pre-partum, and in Pirc rat offspring prior to and during disease progression. Our data show that the gut microbiota varies between rat strains, with LEW/SsNHsd having a greater relative abundance of the bacteria Prevotella copri. The mature gut microbiota of pups resembled the profile of their dams, indicating that the dam is the primary determinant of the developing microbiota. Both male and female F344-Pirc rats harboring the Lewis microbiota had decreased tumor burden relative to genetically identical rats harboring F344 or SD microbiota. Significant negative correlations were detected between tumor burden and the relative abundance of specific taxa from samples taken at weaning and shortly thereafter, prior to observable adenoma development. Notably, this naturally occurring variation in the gut microbiota is associated with a significant difference in severity of colorectal cancer, and the abundance of certain taxa is associated with decreased tumor burden.