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

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.

Efficient and specific generation of knockout mice using Campylobacter jejuni CRISPR/Cas9 system.

The Streptococcus pyogenes CRISPR/Cas9 (SpCas9) system is now widely utilized to generate genome engineered mice; however, some studies raised issues related to off-target mutations with this system. Herein, we utilized the Campylobacter jejuni Cas9 (CjCas9) system to generate knockout mice. We designed sgRNAs targeting mouse Tyr or Foxn1 and microinjected into zygotes along with CjCas9 mRNA. We obtained newborn mice from the microinjected embryos and confirmed that 50% (Tyr) and 38.5% (Foxn1) of the newborn mice have biallelic mutation on the intended target sequences, indicating efficient genome targeting by CjCas9. In addition, we analyzed off-target mutations in founder mutant mice by targeted deep sequencing and whole genome sequencing. Both analyses revealed no off-target mutations at potential off-target sites predicted in silico and no unexpected random mutations in analyzed founder animals. In conclusion, the CjCas9 system can be utilized to generate genome edited mice in a precise manner.

Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency.

The CRISPR/Cas9 system is widely applied in genome engineering due to its simplicity and versatility. Although this has revolutionized genome-editing technology, knockin animal generation via homology directed repair (HDR) is not as efficient as nonhomologous end-joining DNA-repair-dependent knockout. Although its double-strand break activity may vary, Cas9 derived from Streptococcus pyogenens allows robust design of single-guide RNAs (sgRNAs) within the target sequence; However, prescreening for different sgRNA activities delays the process of transgenic animal generation. To overcome this limitation, multiple sets of different sgRNAs were examined for their knockin efficiency. We discovered profound advantages associated with single-stranded oligo-donor-mediated HDR processes using overlapping sgRNAs (sharing at least five base pairs of the target sites) as compared with using non-overlapping sgRNAs for knock-in mouse generation. Studies utilizing cell lines revealed shorter sequence deletions near target mutations using overlapping sgRNAs as compared with those observed using non-overlapping sgRNAs, which may favor the HDR process. Using this simple method, we successfully generated several transgenic mouse lines harboring loxP insertions or single-nucleotide substitutions with a highly efficiency of 18-38%. Our results demonstrate a simple and efficient method for generating transgenic animals harboring foreign-sequence knockins or short-nucleotide substitutions by the use of overlapping sgRNAs.

Neuronal Nitric Oxide Synthase Is a Novel Biomarker for the Interstitial Cells of Cajal in Stress-Induced Diarrhea-Dominant Irritable Bowel Syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder involving changes in normal bowel movements. The pathophysiology of IBS is not clearly understood owing to the lack of identifiable pathological abnormalities and reliable biomarkers. AIM: The aim of this study was to discover the novel and reliable biomarker for IBS. METHOD: In this study, neonatal maternal separation (NMS) stress model was used for the IBS mouse model. Further assessment was conducted with whole gastrointestinal transit test, quantitative RT-PCR, histological examination, and western blot. RESULTS: Male pups developed symptoms similar to those of human IBS with diarrhea (IBS-D), such as low-grade inflammation, stool irregularity, and increased bowel motility. NMS stress influenced to the interstitial cells of Cajal (ICC) and induced altered bowel motility, resulting in IBS-D-like symptoms. In addition, we found neuronal nitric oxide synthase (nNOS) to be a novel biomarker for ICC under NMS stress. nNOS expression was only observed in the ICC of the submucosal plexus of IBS-D mice, and the inhibition of nNOS changed the phenotype from IBS-D to IBS with constipation. CONCLUSION: Our study demonstrates that early-life stress can influence to ICC and modulate bowel activity and that nNOS might be used as a biomarker for ICC stimulation in IBS.

CD80CD86 deficiency disrupts regulatory CD4+FoxP3+T cell homoeostasis and induces autoimmune-like alopecia.

Alopecia areata (AA) is an autoimmune disease that results in spot baldness in humans. Adequate animal models for AA are currently lacking. The objective of this study was to elucidate the mechanism of autoimmune-like alopecia (ALA) in C57BL/6.CD80CD86-deficient (B6.CD80CD86-/- ) mice. Incidence and severity of alopecia were analysed in 58 B6.CD80CD86-/- mice using histological examination, flow cytometry, multiplex enzyme-linked immunosorbent assay, quantitative RT-PCR and CD25 inhibition test. Both male and female B6.CD80CD86-/- mice showed almost 100% incidence of hair loss at 40 weeks of age. Moreover, CD4+FoxP3+Treg (Treg) cell population in B6.CD80CD86-/- mice was significantly lower than in B6 mice, which presumably underlined autoimmune reaction. Histologically, B6.CD80CD86-/- mice showed CD4+ and CD8+ T-cell infiltration around terminal follicle region and exhibited hair follicle destruction in the anagen or catagen stage. Negative correlation between the number of CD4+FoxP3+ Tregs and ALA was confirmed by the CD25 depletion test in B6 mice, as follicle destruction was similar to that observed in B6.CD80CD86-/- animals. CD80CD86 deficiency disrupted CD4+FoxP3+ Treg homoeostasis and prompted the development of ALA. We demonstrated that B6.CD80CD86-/- mice might have several advantages as an ALA model, because they exhibited high incidence of disease phenotype and epipathogenesis similar to that observed in human AA.