Improving Knock In efficiency in mouse generation and Biomarker discovery for Irritable Bowel Syndrome

Abstract

Animal model is an essential tool for studying human disease and assessing newly developing drug. Almost animal model has been developed by chemical induction or genetic modification. The past few years have witnessed remarkable development of sequence-specific DNA endonuclease technologies in model organisms for precise genome editing, which holds the promise to greatly improve the understanding of developmental biology and diseases. Considering cost, design, and efficiency, the CRISPR/Cas9 system has a number of advantages over zinc-finger or TALE protein-fused nucleases. CRISPR/Cas9 system presented high efficiency in knock out (KO), but showed still low efficiency in knock-in (KI)-mediated gene modification. Recently, many studies for improving nuclease-mediated KI efficiency have been reported. Non-homologous end joining (NHEJ) inhibitor such as Scr7 and RS-1 induced 2~4 fold higher frequency, and single strand oligodeoxynucleotide (ssODN) modification with 3 end phosphorothioate or methyl-CpG also showed significant increase of homology direct repair (HDR) efficacy. Different with previous studies, I focus on sgRNA design and inhibitory pathway of homologous recombination (HR) by E3 ubiquitin in G1 stage. Kelch-like ECH-associated protein 1 (KEAP1) is an E3 ubiquitin ligase, which interacts with PALB2. I hypothesized that multiple sgRNAs with similar binding site increase chance of HR, and inhibition of KEAP1 would enhance HR activity in fertile embryo. KEAP1 inhibition showed relatively higher KI efficiency than control (35.7% versus 23.9%) in 110 bp (loxp-multiple cloning sequence-loxp2272) nucleotide insertion into mouse Rosa26 locus, and founder mice with precise KI was produced by microinjection. In further study for generating UPF1 loxp floxed mice, sgRNA with overlapping binding site enhanced about three fold higher KI efficiency than non-overlapped sgRNAs, and KEAP1 inhibition also increased KI rate. Most of all, combination of overlapping sgRNA and KEAP1 inhibition dramatically increase KI rate from 0% to 35.7% in mouse generation which contain single nucleotide polymorphism (cC260T in Morc2a gene). This work demonstrates that overlapping sgRNA and KEAP1 inhibition improve precise KI efficiency in three different experiments, and it would be useful for generating KI animals.

Next, I studied about animal model for human Irritible bowel syndrome (IBS). IBS is a prevalent chronic functional bowel disorder characterized by visceral hyperalgesia, abdominal pain, diarrhea and constipation without any structural cause. Although IBS is not fatal, it worsens to a patients quality of life, and affects approximately 10–20% of worlds population. B6 littermate pups were separated for 3 hours during postnatal days 3-14 or left undisturbed with their dam. Neonatal Maternal Separation (NMS) treatment induced neonatal stress, and it was confirmed by tyrosine hydroxylase (TH) gene expression in adrenal cortex. C57BL/6 mice with NMS showed IBS-D type in water contents analysis (NMS vs control: 14.67% vs 7.65% at 12weeks), but did not show growth retardation. There were no remarkable inflammation lesion in NMS and control mice of every strain in histological examination such as H&E and immunohistochemistry for F4/80, Gr1, CD3 and mast cell chymase. In addition, quantitative PCR for detecting mRNA of Toll-like receptors (TLRs) and inflammatory cytokine genes also did not show significant difference between NMS and control mice. Strong c-kit and nNOS expression was detected in interstitial cell of cajal (ICC) in NMS-treated mice, but control mice showed none or weak c-kit expression. This might indicate that NMS mediated stress induce ICC stimulation, and followed increasing of visceral motor activity. To confirm role of nNOS in NMS-derived IBS model, B6.nNOS-/- was subjected to NMS treatment, and showed opposite results with that of B6. B6 NMS-treated nNOS-/- pups showed IBS-C like phenotype in feces test (NMS vs control: 7.76% vs 11.96% at 18 weeks). C57BL/6 with NMS seemed to be good model for human IBS-D, and nNOS deficient mice with NMS could be useful as IBS-C animal model. The most important finding is high c-kit and nNOS expression in NMS group and it might be hall marker of stress induced ICC stimulation. In conclusion, I successfully established method for high-KI efficiency with overlapping sgRNA, and discover good bio-marker for IBS.