Tissue-specific Genome Editing in vivo by MicroRNA-repressible Anti-CRISPR Proteins [preprint]

CRISPR-Cas systems are bacterial adaptive immune pathways that have revolutionized biotechnology and biomedical applications. Despite the potential for human therapeutic development, there are many hurdles that must be overcome before its use in clinical settings. Some clinical safety concerns arise from persistent activity of Cas9 after the desired editing is complete, or from editing activity in unintended cell types or tissues upon in vivo delivery [e.g. by adeno-associated viruses (AAV)]. Although tissue-specific promoters and serotypes with tissue tropisms can be used, suitably compact promoters are not always available for desired cell types, and AAV tissue tropisms are not absolute. To reinforce tissue-specific editing, we exploited anti-CRISPR proteins (Acrs), which are proteins evolved as countermeasures against CRISPR immunity. To inhibit Cas9 in all ancillary tissues without compromising editing in the target tissue, we established a flexible platform in which an Acr transgene is repressed by endogenous, tissue-specific microRNAs (miRNAs). We demonstrate that miRNAs regulate the expression of an Acr transgene bearing miRNA-binding sites in its 3’ UTR, and control subsequent genome editing outcomes in a cell-type specific manner. We also show that the strategy is applicable to multiple Cas9 orthologs and their respective Acrs. Furthermore, we demonstrate that in vivo delivery of Cas9 and Acrs that are targeted for repression by liver-specific miR-122 allow editing in the liver while Acrs devoid of miRNA regulation prevent Cas9 activity. This strategy provides additional safeguards against off-tissue genome editing by confining Cas9 activity to selected cell types

Tissue-restricted genome editing in vivo specified by microRNA-repressible anti-CRISPR proteins

CRISPR-Cas systems are bacterial adaptive immune pathways that have revolutionized biotechnology and biomedical applications. Despite the potential for human therapeutic development, there are many hurdles that must be overcome before its use in clinical settings. Some clinical safety concerns arise from editing activity in unintended cell types or tissues upon in vivo delivery (e.g., by adeno-associated virus (AAV) vectors). Although tissue-specific promoters and serotypes with tissue tropisms can be used, suitably compact promoters are not always available for desired cell types, and AAV tissue tropism specificities are not absolute. To reinforce tissue-specific editing, we exploited anti-CRISPR proteins (Acrs) that have evolved as natural countermeasures against CRISPR immunity. To inhibit Cas9 in all ancillary tissues without compromising editing in the target tissue, we established a flexible platform in which an Acr transgene is repressed by endogenous, tissue-specific microRNAs (miRNAs). We demonstrate that miRNAs regulate the expression of an Acr transgene bearing miRNA-binding sites in its 3\u27-UTR and control subsequent genome editing outcomes in a cell-type specific manner. We also show that the strategy is applicable to multiple Cas9 orthologs and their respective anti-CRISPRs. Furthermore, we validate this approach in vivo by demonstrating that AAV9 delivery of Nme2Cas9, along with an AcrIIC3 Nme construct that is targeted for repression by liver-specific miR-122, allows editing in the liver while repressing editing in an unintended tissue (heart muscle) in adult mice. This strategy provides safeguards against off-tissue genome editing by confining Cas9 activity to selected cell types

Resveratrol differentially modulates inflammatory responses of microglia and astrocytes

<p>Abstract</p> <p>Background</p> <p>Inflammatory responses in the CNS mediated by activated glial cells play an important role in host-defense but are also involved in the development of neurodegenerative diseases. Resveratrol is a natural polyphenolic compound that has cardioprotective, anticancer and anti-inflammatory properties. We investigated the capacity of resveratrol to protect microglia and astrocyte from inflammatory insults and explored mechanisms underlying different inhibitory effects of resveratrol on microglia and astrocytes.</p> <p>Methods</p> <p>A murine microglia cell line (N9), primary microglia, or astrocytes were stimulated by LPS with or without different concentrations of resveratrol. The expression and release of proinflammatory cytokines (TNF-α, IL-1β, IL-6, MCP-1) and iNOS/NO by the cells were measured by PCR/real-time PCR and ELISA, respectively. The phosphorylation of the MAP kinase superfamily was analyzed by western blotting, and activation of NF-κB and AP-1 was measured by luciferase reporter assay and/or electrophoretic mobility shift assay.</p> <p>Results</p> <p>We found that LPS stimulated the expression of TNF-α, IL-1β, IL-6, MCP-1 and iNOS in murine microglia and astrocytes in which MAP kinases, NF-κB and AP-1 were differentially involved. Resveratrol inhibited LPS-induced expression and release of TNF-α, IL-6, MCP-1, and iNOS/NO in both cell types with more potency in microglia, and inhibited LPS-induced expression of IL-1β in microglia but not astrocytes. Resveratrol had no effect on LPS-stimulated phosphorylation of ERK1/2 and p38 in microglia and astrocytes, but slightly inhibited LPS-stimulated phosphorylation of JNK in astrocytes. Resveratrol inhibited LPS-induced NF-κB activation in both cell types, but inhibited AP-1 activation only in microglia.</p> <p>Conclusion</p> <p>These results suggest that murine microglia and astrocytes produce proinflammatory cytokines and NO in response to LPS in a similar pattern with some differences in signaling molecules involved, and further suggest that resveratrol exerts anti-inflammatory effects in microglia and astrocytes by inhibiting different proinflammatory cytokines and key signaling molecules.</p

Adenovirus-Mediated Somatic Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses

CRISPR/Cas9 derived from the bacterial adaptive immunity pathway is a powerful tool for genome editing, but the safety profiles of in vivo delivered Cas9 (including host immune responses to the bacterial Cas9 protein) have not been comprehensively investigated in model organisms. Nonalcoholic steatohepatitis (NASH) is a prevalent human liver disease characterized by excessive fat accumulation in the liver. In this study, we used adenovirus (Ad) vector to deliver a Streptococcus pyogenes–derived Cas9 system (SpCas9) targeting Pten, a gene involved in NASH and a negative regulator of the PI3K-AKT pathway, in mouse liver. We found that the Ad vector mediated efficient Pten gene editing even in the presence of typical Ad vector-associated immunotoxicity in the liver. Four months after vector infusion, mice receiving the Pten gene-editing Ad vector showed massive hepatomegaly and features of NASH, consistent with the phenotypes following Cre-loxP-induced Pten deficiency in mouse liver. We also detected induction of humoral immunity against SpCas9 and the potential presence of an SpCas9-specific cellular immune response. Our findings provide a strategy to model human liver diseases in mice and highlight the importance considering Cas9-specific immune responses in future translational studies involving in vivo delivery of CRISPR/Cas9

Genetic disruption of oncogenic Kras sensitizes lung cancer cells to Fas receptor-mediated apoptosis

Genetic lesions that activate KRAS account for approximately 30% of the 1.6 million annual cases of lung cancer. Despite clinical need, KRAS is still undruggable using traditional small-molecule drugs/inhibitors. When oncogenic Kras is suppressed by RNA interference, tumors initially regress but eventually recur and proliferate despite suppression of Kras Here, we show that tumor cells can survive knockout of oncogenic Kras, indicating the existence of Kras-independent survival pathways. Thus, even if clinical KRAS inhibitors were available, resistance would remain an obstacle to treatment. Kras-independent cancer cells exhibit decreased colony formation in vitro but retain the ability to form tumors in mice. Comparing the transcriptomes of oncogenic Kras cells and Kras knockout cells, we identified 603 genes that were specifically up-regulated in Kras knockout cells, including the Fas gene, which encodes a cell surface death receptor involved in physiological regulation of apoptosis. Antibodies recognizing Fas receptor efficiently induced apoptosis of Kras knockout cells but not oncogenic Kras-expressing cells. Increased Fas expression in Kras knockout cells was attributed to decreased association of repressive epigenetic marks at the Fas promoter. Concordant with this observation, treating oncogenic Kras cells with histone deacetylase inhibitor and Fas-activating antibody efficiently induced apoptosis, thus bypassing the need to inhibit Kras. Our results suggest that activation of Fas could be exploited as an Achilles\u27 heel in tumors initiated by oncogenic Kras

Depletion of TRRAP induces p53-independent senescence in liver cancer by downregulating mitotic genes

Hepatocellular carcinoma (HCC) is an aggressive subtype of liver cancer with few effective treatments and the underlying mechanisms that drive HCC pathogenesis remain poorly characterized. Identifying genes and pathways essential for HCC cell growth will aid the development of new targeted therapies for HCC. Using a kinome CRISPR screen in three human HCC cell lines, we identified transformation/transcription domain-associated protein (TRRAP) as an essential gene for HCC cell proliferation. TRRAP has been implicated in oncogenic transformation, but how it functions in cancer cell proliferation is not established. Here, we show that depletion of TRRAP or its co-factor, histone acetyltransferase KAT5, inhibits HCC cell growth via induction of p53- and p21-independent senescence. Integrated cancer genomics analyses using patient data and RNA-sequencing identified mitotic genes as key TRRAP/KAT5 targets in HCC, and subsequent cell cycle analyses revealed that TRRAP- and KAT5-depleted cells are arrested at G2/M phase. Depletion of TOP2A, a mitotic gene and TRRAP/KAT5 target, was sufficient to recapitulate the senescent phenotype of TRRAP/KAT5 knockdown. CONCLUSION: Our results uncover a role for TRRAP/KAT5 in promoting HCC cell proliferation via activation of mitotic genes. Targeting the TRRAP/KAT5 complex is a potential therapeutic strategy for HCC

YAP1 withdrawal in hepatoblastoma drives therapeutic differentiation of tumor cells to functional hepatocyte-like cells

BACKGROUND and AIMS: Despite surgical and chemotherapeutic advances, the five-year survival rate for Stage IV Hepatoblastoma (HB), the predominant pediatric liver tumor, remains at 27%. YAP1 and beta-Catenin co-activation occurs in 80% of children\u27s HB; however, a lack of conditional genetic models precludes tumor maintenance exploration. Thus, the need for a targeted therapy remains unmet. Given the predominance of YAP1 and beta-Catenin activation in HB, we sought to evaluate YAP1 as a therapeutic target in HB. APPROACH and RESULTS: We engineered the first conditional HB murine model using hydrodynamic injection to deliver transposon plasmids encoding inducible YAP1(S127A) , constitutive beta-Catenin(DelN90) , and a luciferase reporter to murine liver. Tumor regression was evaluated using bioluminescent imaging, and tumor landscape characterized using RNA and ATAC sequencing, and DNA foot-printing. Here we show that YAP1(S127A) withdrawal mediates \u3e90% tumor regression with survival for 230+ days in mice. YAP1 (S127A) withdrawal promotes apoptosis in a subset of tumor cells and in remaining cells induces a cell fate switch driving therapeutic differentiation of HB tumors into Ki-67 negative hbHep cells with hepatocyte-like morphology and mature hepatocyte gene expression. YAP1 (S127A) withdrawal drives formation of hbHeps by modulating liver differentiation transcription factor (TF) occupancy. Indeed, tumor-derived hbHeps, consistent with their reprogrammed transcriptional landscape, regain partial hepatocyte function and rescue liver damage in mice. CONCLUSIONS: YAP1(S127A) withdrawal, without silencing oncogenic beta-Catenin, significantly regresses hepatoblastoma, providing the first in vivo data to support YAP1 as a therapeutic target for HB. YAP1(S127A) withdrawal alone sufficiently drives long-term regression in hepatoblastoma because it promotes cell death in a subset of tumor cells and modulates transcription factor occupancy to reverse the fate of residual tumor cells to mimic functional hepatocytes

A-MYB/TCFL5 regulatory architecture ensures the production of pachytene piRNAs in placental mammals

In male mice, the transcription factor A-MYB initiates the transcription of pachytene piRNA genes during meiosis. Here, we report that A-MYB activates the transcription factor Tcfl5 produced in pachytene spermatocytes. Subsequently, A-MYB and TCFL5 reciprocally reinforce their own transcription to establish a positive feedback circuit that triggers pachytene piRNA production. TCFL5 regulates the expression of genes required for piRNA maturation and promotes transcription of evolutionarily young pachytene piRNA genes, whereas A-MYB activates the transcription of older pachytene piRNA genes. Intriguingly, pachytene piRNAs from TCFL5-dependent young loci initiate the production of piRNAs from A-MYB-dependent older loci, ensuring the self-propagation of pachytene piRNAs. A-MYB and TCFL5 act via a set of incoherent feedforward loops that drive regulation of gene expression by pachytene piRNAs during spermatogenesis. This regulatory architecture is conserved in rhesus macaque, suggesting that it was present in the last common ancestor of placental mammals

A-MYB/TCFL5 regulatory architecture ensures the production of pachytene piRNAs in placental mammals

In male mice, the transcription factor A MYB initiates the transcription of pachytene piRNA genes during meiosis. Here, we report that A MYB activates the transcription factor Tcfl5 produced in pachytene spermatocytes. Subsequently, A MYB and TCFL5 reciprocally reinforce their own transcription to establish a positive feedback circuit that triggers pachytene piRNA production. TCFL5 regulates the expression of genes required for piRNA maturation and promotes transcription of evolutionarily young pachytene piRNA genes, whereas A-MYB activates the transcription of older pachytene piRNA genes. Intriguingly, pachytene piRNAs from TCFL5-dependent young loci initiates the production of piRNAs from A-MYB-dependent older loci ensuring the self-propagation of pachytene piRNAs. A MYB and TCFL5 act via a set of incoherent feedforward loops that drive regulation of gene expression by pachytene piRNAs during spermatogenesis. This regulatory architecture is conserved in rhesus macaque, suggesting that it was present in the last common ancestor of placental mammals