Precise and Rapid Validation of Candidate Gene by Allele Specific Knockout With CRISPR/Cas9 in Wild Mice.

It is a tempting goal to identify causative genes underlying phenotypic differences among inbred strains of mice, which is a huge reservoir of genetic resources to understand mammalian pathophysiology. In particular, the wild-derived mouse strains harbor enormous genetic variations that have been acquired during evolutionary divergence over 100s of 1000s of years. However, validating the genetic variation in non-classical strains was extremely difficult, until the advent of CRISPR/Cas9 genome editing tools. In this study, we first describe a T cell phenotype in both wild-derived PWD/PhJ parental mice and F1 hybrids, from a cross to C57BL/6 (B6) mice, and we isolate a genetic locus on Chr2, using linkage mapping and chromosome substitution mice. Importantly, we validate the identification of the functional gene controlling this T cell phenotype, Cd44, by allele specific knockout of the PWD copy, leaving the B6 copy completely intact. Our experiments using F1 mice with a dominant phenotype, allowed rapid validation of candidate genes by designing sgRNA PAM sequences that only target the DNA of the PWD genome. We obtained 10 animals derived from B6 eggs fertilized with PWD sperm cells which were subjected to microinjection of CRISPR/Cas9 gene targeting machinery. In the newborns of F1 hybrids, 80% (n = 10) had allele specific knockout of the candidate gene Cd44 of PWD origin, and no mice showed mistargeting of the B6 copy. In the resultant allele-specific knockout F1 mice, we observe full recovery of T cell phenotype. Therefore, our study provided a precise and rapid approach to functionally validate genes that could facilitate gene discovery in classic mouse genetics. More importantly, as we succeeded in genetic manipulation of mice, allele specific knockout could provide the possibility to inactivate disease alleles while keeping the normal allele of the gene intact in human cells.

A severe atherosclerosis mouse model on the resistant NOD background.

Atherosclerosis is a complex disease affecting arterial blood vessels and blood flow that could result in a variety of life-threatening consequences. Disease models with diverged genomes are necessary for understanding the genetic architecture of this complex disease. Non-obese diabetic (NOD) mice are highly polymorphic and widely used for studies of type 1 diabetes and autoimmunity. Understanding atherosclerosis development in the NOD strain is of particular interest as human atherosclerosis on the diabetic and autoimmune background has not been successfully modeled. In this study, we used CRISPR/Cas9 genome editing to genetically disrupt apolipoprotein E (ApoE) and low-density lipoprotein receptor (LDLR) expression on the pure NOD background, and compared phenotype between single-gene-deleted mice and double-knockout mutants with reference to ApoE-deficient C57BL/6 mice. We found that genetic ablation of Ldlr or Apoe in NOD mice was not sufficient to establish an atherosclerosis model, in contrast to ApoE-deficient C57BL/6 mice fed a high-fat diet (HFD) for over 12 weeks. We further obtained NOD mice deficient in both LDLR and ApoE, and assessed the severity of atherosclerosis and immune response to hyperlipidemia in comparison to ApoE-deficient C57BL/6 mice. Strikingly, the double-knockout NOD mice treated with a HFD developed severe atherosclerosis with aorta narrowed by over 60% by plaques, accompanied by destruction of pancreatic islets and an inflammatory response to hyperlipidemia. Therefore, we succeeded in obtaining a genetic model with severe atherosclerosis on the NOD background, which is highly resistant to the disease. This model is useful for the study of atherosclerosis in the setting of autoimmunity.

Speed genome editing by transient CRISPR/Cas9 targeting and large DNA fragment deletion.

Genetic engineering of cell lines and model organisms has been facilitated enormously by the CRISPR/Cas9 system. However, in cell lines it remains labor intensive and time consuming to obtain desirable mutant clones due to the difficulties in isolating the mutated clones and sophisticated genotyping. In this study, we have validated fluorescent protein reporter aided cell sorting which enables the isolation of maximal diversity in mutant cells. We further applied two spectrally distinct fluorescent proteins DsRed2 and ECFP as reporters for independent CRISPR/Cas9 mediated targeting, which allows for one-cell-one-well sorting of the mutant cells. Because of ultra-high efficiency of the CRISPR/Cas9 system with dual reporters and large DNA fragment deletion resulting from independent loci cleavage, monoclonal mutant cells could be easily identified by conventional PCR. In the speed genome editing method presented here, sophisticated genotyping methods are not necessary to identify loss of function mutations after CRISPR/Cas9 genome editing, and desirable loss of function mutant clones could be obtained in less than one month following transfection.

Silencing of the glypican-3 gene affects the biological behavior of human hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death in the world. The gene glypican-3 (GPC3) is reported to be a potential therapeutic target for HCC. In this study, we use RNA interference with lentiviral vectors to explore the effect of GPC3 silencing on the biological behavior of HCC cells and the potential role of the GPC3 protein in the activation of epithelial-mesenchymal transition (EMT), which relates to HCC cell invasion and migration. Our data suggest that GPC3 silencing leads to a decrease in HCC cell proliferation and to an increase in apoptosis. We demonstrated that GPC3 silencing regulates cell invasion and migration, most probably through the activation of the EMT cellular program. In conclusion, GPC3 is associated with the HCC cell biological behavior, while the relationship between GPC3 and EMT in tumorigenesis of HCC deserves future investigation.

Stable knockdown of protein kinase CK2-alpha (CK2α) inhibits migration and invasion and induces inactivation of hedgehog signaling pathway in hepatocellular carcinoma Hep G2 cells.

Protein kinase CK2-alpha (CK2α), one isoform of the catalytic subunits of serine/threonine kinase CK2, has been indicated to participate in tumorigenesis of various malignancies, including hepatocellular carcinoma (HCC). In the present study, in order to explore the potential role of CK2α in human HCC, we employed short hairpin RNA (shRNA)-mediated RNA interference (RNAi) technology to inhibit the endogenous CK2α expression in HCC cells and established a Hep G2 cell line with stable knockdown of CK2α. Results from wound healing and transwell invasion assays indicated that stable knockdown of CK2α markedly inhibited Hep G2 cell migration and invasion as compared with those transfected with a negative control plasmid. This alteration was accompanied with expression down-regulation of matrix metalloproteinase (MMP)-2, MMP-9, Snail, Slug, Vimentin, and up-regulation of epithelial cadherin (E-cadherin). Moreover, CK2α silencing also induced inactivation of Hedgehog signaling pathway by inhibiting Gli1 and Patched homolog 1 (PTCH1) expressions in HCC cells. Collectively, these results demonstrate that knockdown of CK2α can suppress cell migration and invasion, reduces expression of MMPs, inhibits epithelial-mesenchymal transition (EMT) process and induces inactivation of Hedgehog pathway in HCC cells in vitro. Our study provides in vitro evidence to demonstrate that the pathogenesis of human HCC may be correlated with the high expression of CK2α.