gene-knock-crisprcas9-cell-sorting-macrophage-t-cell

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

In this study, we performed ENU mutagenesis and multi-parameter flow cytometric analysis in C57BL/6 mice to uncover novel genes or alleles regulating immune cell development. We identified a novel mutant allele of Cd4 gene which completely blocked development of a major subset of T cells named CD4 T cell. Our data for the first time showed experimentally in mice the critical role of the first extracellular domain, by obtaining mice with a loss of function mutation from Ile to Asn at the position 99 of CD4 (I99N). Interestingly, such CD4 mutant protein can be expressed on the surface of human cells, and the mRNA stability could be also affected by this point mutation, suggesting that absence of CD4 T cells in mice rooted in the deficiency in function and expression of CD4. In addition, we used this novel CD4 T cell deficient model as recipient mice for adoptive transfer experiment, and showed that it could be an optimal model for study of CD4 T cells.

A point mutation in the extracellular domain of CD4 completely abolishes CD4 T cell development in C57BL/6 mouse.

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.

The activation of T cells by the T cell antigen receptor (TCR) results in the formation of signaling protein complexes (signalosomes), the composition of which has not been analyzed at a systems level. Here, we isolated primary CD4 T cells from 15 gene-targeted mice, each expressing one tagged form of a canonical protein of the TCR-signaling pathway. Using affinity purification coupled with mass spectrometry, we analyzed the composition and dynamics of the signalosomes assembling around each of the tagged proteins over 600 s of TCR engagement. We showed that the TCR signal-transduction network comprises at least 277 unique proteins involved in 366 high-confidence interactions, and that TCR signals diversify extensively at the level of the plasma membrane. Integrating the cellular abundance of the interacting proteins and their interaction stoichiometry provided a quantitative and contextual view of each documented interaction, permitting anticipation of whether ablation of a single interacting protein can impinge on the whole TCR signal-transduction network.

Quantitative interactomics in primary T cells unveils TCR signal diversification extent and dynamics.

Extracellular matrix mineralization is critical for osteogenesis, and its dysregulation could result in osteoporosis and vascular calcification. IKK/NF-κB activation inhibits differentiation of osteoblasts, and reduces extracellular matrix mineralization, however the underlying mechanisms are poorly understood. In this study, we used CRISPR/Cas9 system to permanently inactivate IKKβ in preosteoblast cells and confirmed that such cells displayed dramatic increase in extracellular matrix mineralization associated with JNK phosphorylation. Such observation was also found in our study using IKKβ-deficient primary murine osteoblasts. Interestingly, we found that in     or     double knockout cells, the enhanced mineralization caused by IKKβ deficiency was completely abolished, and deletion of either  or  was sufficient to dampen c-Jun phosphorylation. In further experiments, we discovered that absence of JNK1 or JNK2 on IKKβ-deficient background resulted in highly conserved transcriptomic alteration in response to osteogenic induction. Therefore, identification of the indispensable roles of JNK1 and JNK2 in activating c-Jun and promoting osteoblast differentiation on IKKβ-deficient background provided novel insights into restoring homeostasis in extracellular matrix mineralization.

Both JNK1 and JNK2 Are Indispensable for Sensitized Extracellular Matrix Mineralization in IKKβ-Deficient Osteoblasts.

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.

Schistosomiasis is among the major neglected tropical diseases and effective prevention by boosting the immune system is still not available. T cells are key cellular components governing adaptive immune response to various infections. While common laboratory mice, such as C57BL/6, are highly susceptible to schistosomiasis, the SD rats are extremely resistant. However, whether adaptive immunity is necessary for such natural resistance to schistosomiasis in rats remains to be determined. Therefore, it is necessary to establish genetic model deficient in T cells and adaptive immunity on the resistant SD background, and to characterize liver pathology during schistosomiasis. In this study we compared experimental schistosomiasis in highly susceptible C57BL/6 (B6) mice and in resistant SD rats, using cercariae of Schistosoma japonicum. We observed a marked T cell expansion in the spleen of infected B6 mice, but not resistant SD rats. Interestingly, CD3e-/- B6 mice in which T cells are completely absent, the infectious burden of adult worms was significantly higher than that in WT mice, suggesting an anti-parasitic role for T cells in B6 mice during schistosome infection. In further experiments, we established Lck deficient SD rats by using CRISPR/Cas9 in which T cell development was completely abolished. Strikingly, we found that such Lck deficiency in SD rats severely impaired their natural resistance to schistosome infection, and fostered parasite growth. Together with an additional genetic model deficient in T cells, the CD3e-/- SD rats, we confirmed the absence of T cell resulted in loss of natural resistance to schistosome infection, but also mitigated liver immunopathology. Our further experiments showed that regulatory T cell differentiation in infected SD rats was significantly decreased during schistosomiasis, in contrast to significant increase of regulatory T cells in infected B6 mice. These data suggest that T cell mediated immune tolerance facilitates persistent infection in mice but not in SD rats. The demonstration of an important role for T cells in natural resistance of SD rats to schistosomiasis provides experimental evidences supporting the rationale to boost T cell responses in humans to prevent and treat schistosomiasis.

Loss of natural resistance to schistosome in T cell deficient rat.

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 homeostasis of protein palmitoylation and depalmitoylation is essential for proper physiological functions in various tissues, in particular the central nervous system (CNS). The dysfunction of PPT1 (PPT1-KI, infantile neuronal ceroid lipofuscinosis [INCL] mouse model), which catalyze the depalmitoylation process, results in serious neurodegeneration accompanied by severe astrogliosis in the brain. Endeavoring to determine critical factors that might account for the pathogenesis in CNS by palm-proteomics, glial fibrillary acidic protein (GFAP) was spotted, indicating that GFAP is probably palmitoylated. Questions concerning if GFAP is indeed palmitoylated in vivo and how palmitoylation of GFAP might participate in neural pathology remain unexplored and are waiting to be investigated. Here we show that GFAP is readily palmitoylated in vitro and in vivo; specifically, cysteine-291 is the unique palmitoylated residue in GFAP. Interestingly, it was found that palmitoylated GFAP promotes astrocyte proliferation in vitro. Furthermore, we showed that PPT1 depalmitoylates GFAP, and the level of palmitoylated GFAP is overwhelmingly up-regulated in PPT1-knockin mice, which lead us to speculate that the elevated level of palmitoylated GFAP might accelerate astrocyte proliferation in vivo and ultimately led to astrogliosis in INCL. Indeed, blocking palmitoylation by mutating cysteine-291 into alanine in GFAP attenuate astrogliosis, and remarkably, the concurrent neurodegenerative pathology in PPT1-knockin mice. Together, these findings demonstrate that hyperpalmitoylated GFAP plays critical roles in regulating the pathogenesis of astrogliosis and neurodegeneration in the CNS, and most importantly, pinpointing that cysteine-291 in GFAP might be a valuable pharmaceutical target for treating INCL and other potential neurodegenerative diseases.

GFAP hyperpalmitoylation exacerbates astrogliosis and neurodegenerative pathology in PPT1-deficient mice.

Atherosclerosis, a progressive inflammatory disease characterized by elevated inflammation and lipid accumulation in the aortic endothelium, arises in part from the infiltration of inflammatory cells into the vascular wall. However, it is not fully defined how inflammatory cells, especially macrophages, affect the pathogenesis of atherosclerosis. Schlafen4 (Slfn4) mRNA is remarkably upregulated upon ox-LDL stimulation in macrophages. Nonetheless, the role of Slfn4 in foam cell formation remains unclear.

Slfn4 deficiency improves MAPK-mediated inflammation, oxidative stress, apoptosis and abates atherosclerosis progression in apolipoprotein E-deficient mice.

Article	Year
A point mutation in the extracellular domain of CD4 completely abolishes CD4 T cell development in C57BL/6 mouse. Molecular immunology\| PubMed ID: 29028486	2017
Speed genome editing by transient CRISPR/Cas9 targeting and large DNA fragment deletion. Journal of biotechnology\| PubMed ID: 29886029	2018
A severe atherosclerosis mouse model on the resistant NOD background. Disease models & mechanisms\| PubMed ID: 30305306	2018
Precise and Rapid Validation of Candidate Gene by Allele Specific Knockout With CRISPR/Cas9 in Wild Mice. Frontiers in genetics\| PubMed ID: 30838037	2019
The three members of the Vav family proteins form complexes that concur to foam cell formation and atherosclerosis. Journal of lipid research\| PubMed ID: 31570505	2019
Quantitative interactomics in primary T cells unveils TCR signal diversification extent and dynamics. Nature immunology\| PubMed ID: 31591574	2019
Both JNK1 and JNK2 Are Indispensable for Sensitized Extracellular Matrix Mineralization in IKKβ-Deficient Osteoblasts. Frontiers in endocrinology\| PubMed ID: 32117051	2020
Gfi1 and Zc3h12c orchestrate a negative feedback loop that inhibits NF-kB activation during inflammation in macrophages. Molecular immunology\| PubMed ID: 33157351	2020
Concomitant expression of inhibitory molecules for T cell activation predicts poor survival in patients with esophageal squamous cell carcinoma. Current cancer drug targets\| PubMed ID: 33222673	2020
Loss of natural resistance to schistosome in T cell deficient rat. PLoS neglected tropical diseases\| PubMed ID: 33347431	2020
An inducible model for specific neutrophil depletion by diphtheria toxin in mice. Science China. Life sciences\| PubMed ID: 33420927	2021
Zdhhc2 Is Essential for Plasmacytoid Dendritic Cells Mediated Inflammatory Response in Psoriasis. Frontiers in immunology\| PubMed ID: 33488612	2020
GFAP hyperpalmitoylation exacerbates astrogliosis and neurodegenerative pathology in PPT1-deficient mice. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 33753498	2021
Slfn4 deficiency improves MAPK-mediated inflammation, oxidative stress, apoptosis and abates atherosclerosis progression in apolipoprotein E-deficient mice. Atherosclerosis\| PubMed ID: 34757313	2021

Liaoxun Lu

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