Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...

Xinxiang medical university

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.

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

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  or  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.

A severe atherosclerosis mouse model on the resistant NOD background.

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, , 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% ( = 10) had allele specific knockout of the candidate gene  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.

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

During foam cell formation and atherosclerosis development, the scavenger receptor CD36 plays critical roles in lipid uptake and triggering of atherogenicity via the activation of Vav molecules. The Vav family includes three highly conserved members known as Vav1, Vav2, and Vav3. As Vav1 and Vav3 were found to exert function in atherosclerosis development, it remains thus to decipher whether Vav2 also plays a role in the development of atherosclerosis. In this study we found that Vav2 deficiency in RAW264.7 macrophages significantly diminished oxidized LDL uptake and CD36 signaling, demonstrating that each Vav protein family member was required for foam cell formation. Genetic disruption of Vav2 in ApoE-deficient C57BL/6 mice significantly inhibited the severity of atherosclerosis. Strikingly, we further found that the genetic deletion of each member of the Vav protein family by CRISPR/Cas9 resulted in a similar alteration of transcriptomic profiles of macrophages. The three members of the Vav proteins were found to form complexes, and genetic ablation of each single Vav molecule was sufficient to prevent endocytosis of CD36. The functional interdependence of the three Vav family members in foam cell formation was due to their indispensable roles in transcriptomic programing, lipid uptake, and activation of the JNK kinase in macrophages.

The three members of the Vav family proteins form complexes that concur to foam cell formation and atherosclerosis.

Many mutations in the signal peptide and propeptide of factor IX (FIX) cause hemophilia B. A FIX variants database reports 28 unique missense mutations in these regions that lead to FIX deficiency, but the underlying mechanism is known only for the mutations on R43 that interfere with propeptide cleavage. It remains unclear how other mutations result in FIX deficiency and why patients carrying the same mutation have different bleeding tendencies. Here, we modify a cell-based reporter assay to characterize the missense mutations in the signal peptide and propeptide of FIX. The results show that the level of secreted conformation-specific reporter (SCSR), which has a functional γ-carboxyglutamate (Gla) domain of FIX, decreases significantly in most mutations. The decreased SCSR level is consistent with FIX deficiency in hemophilia B patients. Moreover, we find that the decrease in the SCSR level is caused by several distinct mechanisms, including interfering with cotranslational translocation into the endoplasmic reticulum, protein secretion, γ-carboxylation of the Gla domain, and cleavage of the signal peptide or propeptide. Importantly, our results also show that the SCSR levels of most signal peptide and propeptide mutations increase with vitamin K concentration, suggesting that the heterogeneity of bleeding tendencies may be related to vitamin K levels in the body. Thus, oral administration of vitamin K may alleviate the severity of bleeding tendencies in patients with missense mutations in the FIX signal peptide and propeptide regions.

Characterization of missense mutations in the signal peptide and propeptide of FIX in hemophilia B by a cell-based assay.

NF-κB activation is essential in mediating the induction of pro-inflammatory cytokines and also plays a key role in regulating the inflammatory response through intricate mechanisms. In this study, loss of Gfi1 was found to be associated with transcriptomic profiles related to NF-κB activation, including an increase in pro-inflammatory cytokines. Genetically inactivating the IKK/NF-κB signaling pathway in macrophages showed that Gfi1 deficiency led to pro-inflammatory cytokine production requiring NF-κB activation. More importantly, we revealed that one of the under-researched mechanisms, involving Gfi1 and Zc3h12c exerted negative regulation on NF-κB activation. Both Gfi1 and Zc3h12c were found to inhibit NF-κB activation, and double knockout exhibited additive roles of Gfi1 and Zc3h12c in preventing proinflammatory cytokine production. The loss of Gfi1 upregulated Zc3h12c which in turn inhibited NF-κB activation. Therefore, this study delineates the function of Zc3h12c in enhancing the negative regulation of Gfi1 through NF-κB activation during inflammation in macrophages.

Gfi1 and Zc3h12c orchestrate a negative feedback loop that inhibits NF-kB activation during inflammation in macrophages.

Esophageal squamous cell carcinoma (ESCC) is a major subtype of esophageal cancers. The Five-year survival rate of ESCC is low and molecular targets for ESCC treatment and prognosis assessment are very limited. T cells are critical for clearance of cancer cells and blockade of co-inhibitory molecules for T cell activation has emerged as a promising therapy to treat cancer patients. However, in ESCC patients such co-inhibitory molecules regulating T cell activation is poorly documented.

Concomitant expression of inhibitory molecules for T cell activation predicts poor survival in patients with esophageal squamous cell carcinoma.

Neutrophils are crucial for immunity and play important roles in inflammatory diseases; however, mouse models selectively deficient in neutrophils are limited, and neutrophil-specific diphtheria toxin (DT)-based depletion system has not yet been established. In this study, we generated a novel knock-in mouse model expressing diphtheria toxin receptor (DTR) under control of the endogenous Ly6G promoter. We showed that DTR expression was restricted to Ly6G neutrophils and complete depletion of neutrophils could be achieved by DT treatment at 24-48 h intervals. We characterized the effects of specific neutrophil depletion in mice at steady-state, with acute inflammation and during tumor growth. Our study presents a valuable new tool to study the roles of neutrophils in the immune system and during tumor progression.

An inducible model for specific neutrophil depletion by diphtheria toxin in mice.

Zdhhc family genes are composed of 24 members that regulate palmitoylation, a post-translational modification process for proteins. Mutations in genes that alter palmitoylation or de-palmitoylation could result in neurodegenerative diseases and inflammatory disorders. In this study, we found that Zdhhc2 was robustly induced in psoriatic skin and loss of Zdhhc2 in mice by CRISPR/Cas9 dramatically inhibited pathology of the ear skin following imiquimod treatment. As psoriasis is an inflammatory disorder, we analyzed tissue infiltrating immune cells and cytokine production. Strikingly we found that a master psoriatic cytokine interferon-α (IFN-α) in the lesioned skin of wildtype (WT) mice was 23-fold higher than that in Zdhhc2 deficient counterparts. In addition, we found that CD45 white blood cells (WBC) infiltrating in the skin of Zdhhc2 deficient mice were also significantly reduced. Amelioration in psoriasis and dramatically reduced inflammation of Zdhhc2 deficient mice led us to analyze the cellular components that were affected by loss of Zdhhc2. We found that imiquimod induced plasmacytoid dendritic cell (pDC) accumulation in psoriatic skin, spleen, and draining lymph nodes (DLN) were drastically decreased in Zdhhc2 deficient mice, and the expression of pDC activation marker CD80 also exhibited significantly inhibited in psoriatic skin. In further experiments, we confirmed the cell intrinsic effect of Zdhhc2 on pDCs as we found that loss of zDHHC2 in human CAL-1 pDC dampened both interferon regulatory factor 7 (IRF7) phosphorylation and IFN-α production. Therefore, we identified novel function of Zdhhc2 in controlling inflammatory response in psoriasis in mice and we also confirmed that crucial role of Zdhhc2 in pDCs by regulating IRF7 activity and production of the critical cytokine. Our results finding the dependence of IFN-α production on Zdhhc2 in inflamed murine skin and in human pDCs provide rationale for targeting this new molecule in treatment of inflammation.

Zdhhc2 Is Essential for Plasmacytoid Dendritic Cells Mediated Inflammatory Response in Psoriasis.

The mammalian target of rapamycin complex 1 (mTORC1) is a crucial regulator of adipogenesis and systemic energy metabolism. Its dysregulation leads to a diversity of metabolic diseases, including obesity and type 2 diabetes. DEP-domain containing 5 (DEPDC5) is a critical component of GATOR1 complex that functions as a key inhibitor of mTORC1. So far, its function in adipose tissue remains largely unknown. Herein we evaluated how persistent mTORC1 activation in adipocyte via Depdc5 knockout modulates adiposity in vivo. Our data indicated that adipocyte-specific knockout of Depdc5 in aged mice led to reduced visceral fat, aggravated insulin resistance and enhanced adipose tissue inflammation. Moreover, we found that Depdc5 ablation resulted in upregulation of adipose triglyceride lipase (ATGL) in adipocytes and elevated levels of serum free fatty acids (FFAs). Intriguingly, rapamycin treatment did not reverse insulin resistance but alleviated adipose tissue inflammation caused by Depdc5 deletion. Taken together, our findings revealed that mTORC1 activation caused by Depdc5 deletion promotes lipolysis process and further exacerbates insulin resistance and adipose tissue inflammation in mice.

Adipocyte-specific deletion of Depdc5 exacerbates insulin resistance and adipose tissue inflammation in mice.

The mechanistic target of rapamycin complex 1 (mTORC1) acts as a central regulator of metabolic pathways that drive cellular growth. Abnormal activation of mTORC1 occurs at high frequency in human and mouse hepatocellular carcinoma (HCC). DEP domain-containing protein 5 (DEPDC5), a component of GATOR1 complex, is a repressor of amino acid-sensing branch of the mTORC1 pathway. In the current study, we found that persistent activation of hepatic mTORC1 signaling caused by Depdc5 ablation was sufficient to induce a pathological program of liver damage, inflammation and fibrosis that triggers spontaneous HCC development. Take advantage of the combinatory treatment with a single dose of diethylnitrosamine (DEN) and chronic feeding with high-fat diet (HFD), we demonstrated that hepatic depdc5 deletion did not aggravate DEN&HFD induced liver tumorigenesis, probably due to its protective effects on diet-induced liver steatosis. In addition, we further showed that chronic rapamycin treatment did not have any apparent tumor-suppressing effects on DEN&HFD treated control mice, whereas it dramatically reduced the tumor burden in mice with hepatic Depdc5 ablation. This study provides the novel in vivo evidence for Depdc5 deletion mediated mTORC1 hyperactivation in liver tumorigenesis caused by aging or DEN&HFD treatment. Moreover, our findings also propose that pharmacological inhibition of mTORC1 signaling maybe a promising strategy to treat HCC patients with mutations in DEPDC5 gene.

Persistent mTORC1 activation via Depdc5 deletion results in spontaneous hepatocellular carcinoma but does not exacerbate carcinogen- and high-fat diet-induced hepatic carcinogenesis in mice.

Rong Huang

The Laboratory of Genetic Regulators in the Immune System,
School of Laboratory Medicine,
The Laboratory of Genetic Regulators in the Immune System, School of Laboratory Medicine

Xinxiang medical university

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Rong Huang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}The Laboratory of Genetic Regulators in the Immune System,School of Laboratory Medicine,The Laboratory of Genetic Regulators in the Immune System, School of Laboratory Medicine

Xinxiang medical university

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

The Laboratory of Genetic Regulators in the Immune System,
School of Laboratory Medicine,
The Laboratory of Genetic Regulators in the Immune System, School of Laboratory Medicine