CRISPR Activation System Research Articles

CRISPR activation identifies a novel miR-2861 binding site that facilitates the osteogenesis of human mesenchymal stem cells.

We investigated the regulation of histone deacetylases (HDACs) by miR-2861 in the osteoblastic differentiation of human mesenchymal stem cells (MSCs) and miR-2861 binding site by CRISPR activation (CRISPRa). Transfection of miR-2861 into human MSCs was performed and the effect on osteoblast differentiation was analyzed. Using catalytically inactive Cas12a, the CRISPRa system induced targeted overexpression of endogenous miRNA and repressed the luciferase activities of reporters that contained functional miRNA target sites. The delivery of miR-2861 into MSCs enhanced osteoblast differentiation by decreased expressions of the HDAC1, 4 and 5 genes. The mechanism of HDAC5 repression by miR-2861 in humans has not been fully elucidated. To this end, the HDAC5 mRNA sequence was analyzed and a putative primate-specific miR-2861 binding site was identified in the 3' untranslated region (3'-UTR). CRISPRa was applied to validate the putative binding site and an increase in endogenous miR-2861 was found to repress the expression of a reporter that contained the novel miR-2861 binding site. The delivery of miR-2861 to human MSCs enhanced osteoblast differentiation. In the 3'-UTR, the HDAC5 repression was mediated by the miR-2861 binding site, and miR-2861 promoted osteoblast differentiation via the inhibition of HDAC5 through a primate-specific miRNA binding site. Therefore, miRNAmiR-2861 with the CRISPRa methods might be a good biomaterial for osteogenesis augmentation.

Activating UCHL1 through the CRISPR activation system promotes cartilage differentiation mediated by HIF-1α/SOX9.

Developing strategies to enhance cartilage differentiation in mesenchymal stem cells and preserve the extracellular matrix is crucial for successful cartilage tissue reconstruction. Hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in maintaining the extracellular matrix and chondrocyte phenotype, thus serving as a key regulator in chondral tissue engineering strategies. Recent studies have shown that Ubiquitin C-terminal hydrolase L1 (UCHL1) is involved in the deubiquitylation of HIF-1α. However, the regulatory role of UCHL1 in chondrogenic differentiation has not been investigated. In the present study, we initially validated the promotive effect of UCHL1 expression on chondrogenesis in adipose-derived stem cells (ADSCs). Subsequently, a hybrid baculovirus system was designed and employed to utilize three CRISPR activation (CRISPRa) systems, employing dead Cas9 (dCas9) from three distinct bacterial sources to target UCHL1. Then UCHL1 and HIF-1α inhibitor and siRNA targeting SRY-box transcription factor 9 (SOX9) were used to block UCHL1, HIF-1α and SOX9, respectively. Cartilage differentiation and chondrogenesis were measured by qRT-PCR, immunofluorescence and histological staining. We observed that the CRISPRa system derived from Staphylococcus aureus exhibited superior efficiency in activating UCHL1 compared to the commonly used the CRISPRa system derived from Streptococcus pyogenes. Furthermore, the duration of activation was extended by utilizing the Cre/loxP-based hybrid baculovirus. Moreover, our findings show that UCHL1 enhances SOX9 expression by regulating the stability and localization of HIF-1α, which promotes cartilage production in ADSCs. These findings suggest that activating UCHL1 using the CRISPRa system holds significant potential for applications in cartilage regeneration.

A Nanozyme-Boosted MOF-CRISPR Platform for Treatment of Alzheimer's Disease.

Rectifying the aberrant microenvironment of a disease through maintenance of redox homeostasis has emerged as a promising perspective with significant therapeutic potential for Alzheimer's disease (AD). Herein, we design and construct a novel nanozyme-boosted MOF-CRISPR platform (CMOPKP), which can maintain redox homeostasis and rescue the impaired microenvironment of AD. By modifying the targeted peptides KLVFFAED, CMOPKP can traverse the blood-brain barrier and deliver the CRISPR activation system for precise activation of the Nrf2 signaling pathway and downstream redox proteins in regions characterized by oxidative stress, thereby reinstating neuronal antioxidant capacity and preserving redox homeostasis. Furthermore, cerium dioxide possessing catalase enzyme-like activity can synergistically alleviate oxidative stress. Further in vivo studies demonstrate that CMOPKP can effectively alleviate cognitive impairment in 3xTg-AD mouse models. Therefore, our design presents an effective way for regulating redox homeostasis in AD, which shows promise as a therapeutic strategy for mitigating oxidative stress in AD.

Identification of cis-acting elements that regulate expression of the progesterone receptor gene PGR in the uterus.

The steroid hormone progesterone regulates a wide range of functions in the uterus, including menstruation, pregnancy establishment, pregnancy maintenance, and labor. The cellular effects of progesterone are mediated by the nuclear progesterone receptor (PGR). Progesterone resistance, a decreased cellular response to progesterone,is implicated in a wide range of pathologies including endometriosis, preterm birth, and endometrial cancer. Up to 10% of females within reproductive age are impacted by endometriosis, and no cure is available. This study hypothesizes that misregulation of PGR by upstream regulators is a potential cause of progesterone resistance. However, much remains unknown about the regulation of PGR expression, including the location of enhancers and the identity of transcriptional regulators that control PGR expression. In this study, candidate regulatory elements of PGR were identified by integrative analysis of genomic data, and their function was tested using the CRISPR activation system, with PGR mRNA levels as the readout. Using these methods, two PGR enhancers located at the PGR 3’ untranslated region and 200 kbs upstream of the PGR transcription start site were identified. These enhancers permitted the identification of candidate upstream regulators of PGR expression in the uterus, including GATA2, ARID1A, KLF9, and ESR1. Activation of ESR1 increased PGR mRNA levels in the presence of estradiol in cultured uterine cells. Through the discovery of PGR regulators, we can improve our understanding of molecular mechanisms of progesterone resistance. Ultimately, these regulators could be evaluated in cases of progesterone resistance and targeted in the development of therapies.

Efficient activation of hundreds of LTR12C elements reveals cis-regulatory function determined by distinct epigenetic mechanisms.

Long terminal repeats (LTRs), which often contain promoter and enhancer sequences of intact endogenous retroviruses (ERVs), are known to be co-opted as cis-regulatory elements for fine-tuning host-coding gene expression. Since LTRs are mainly silenced by the deposition of repressive epigenetic marks, substantial activation of LTRs has been found in human cells after treatment with epigenetic inhibitors. Although the LTR12C family makes up the majority of ERVs activated by epigenetic inhibitors, how these epigenetically and transcriptionally activated LTR12C elements can regulate the host-coding gene expression remains unclear due to genome-wide alteration of transcriptional changes after epigenetic inhibitor treatments. Here, we specifically transactivated >600 LTR12C elements by using single guide RNA-based dCas9-SunTag-VP64, a site-specific targeting CRISPR activation (CRISPRa)system, with minimal off-target events. Interestingly, most of the transactivated LTR12C elements acquired the H3K27ac-marked enhancer feature, while only 20% were co-marked with promoter-associated H3K4me3 modifications. The enrichment of the H3K4me3 signal was intricately associated with downstream regions of LTR12C, such as internal regions of intact ERV9 or other types of retrotransposons. Here, we leverage an optimized CRISPRa system to identify two distinct epigenetic signatures that define LTR12C transcriptional activation, which modulate the expression of proximal protein-coding genes.

Distinct roles of Bdnf I and Bdnf IV transcript variant expression in hippocampal neurons.

Brain-derived neurotrophic factor (Bdnf) plays a critical role in brain development, dendritic growth, synaptic plasticity, as well as learning and memory. The rodent Bdnf gene contains nine 5' non-coding exons (I-IXa), which are spliced to a common 3' coding exon (IX). Transcription of individual Bdnf variants, which all encode the same BDNF protein, is initiated at unique promoters upstream of each non-coding exon, enabling precise spatiotemporal and activity-dependent regulation of Bdnf expression. Although prior evidence suggests that Bdnf transcripts containing exon I (Bdnf I) or exon IV (Bdnf IV) are uniquely regulated by neuronal activity, the functional significance of different Bdnf transcript variants remains unclear. To investigate functional roles of activity-dependent Bdnf I and IV transcripts, we used a CRISPR activation system in which catalytically dead Cas9 fused to a transcriptional activator (VPR) is targeted to individual Bdnf promoters with single guide RNAs, resulting in transcript-specific Bdnf upregulation. Bdnf I upregulation is associated with gene expression changes linked to dendritic growth, while Bdnf IV upregulation is associated with genes that regulate protein catabolism. Upregulation of Bdnf I, but not Bdnf IV, increased mushroom spine density, volume, length, and head diameter, and also produced more complex dendritic arbors in cultured rat hippocampal neurons. In contrast, upregulation of Bdnf IV, but not Bdnf I, in the rat hippocampus attenuated contextual fear expression. Our data suggest that while Bdnf I and IV are both activity-dependent, BDNF produced from these promoters may serve unique cellular, synaptic, and behavioral functions.

An inducible CRISPR activation tool for accelerating plant regeneration

The inducible CRISPR activation (CRISPR-a) system offers unparalleled precision and versatility for regulating endogenous genes, making it highly sought after in plant research. In this study, we developed a chemically inducible CRISPR-a tool for plants called ER-Tag by combining the LexA-VP16-ER inducible system with the SunTag CRISPR-a system. We systematically compared different induction strategies and achieved high efficiency in target gene activation. We demonstrated that guide RNAs can be multiplexed and pooled for large-scale screening of effective morphogenic genes and gene pairs involved in plant regeneration. Further experiments showed that induced activation of these morphogenic genes can accelerate regeneration and improve regeneration efficiency in both eudicot and monocot plants, including alfalfa, woodland strawberry, and sheepgrass. Our study expands the CRISPR toolset in plants and provides a powerful new strategy for studying gene function when constitutive expression is not feasible or ideal.

Modulating the tumoral SPARC content to enhance albumin-based drug delivery for cancer therapy

Insufficient delivery of therapeutic agents into solid tumors by systemic administration remains a major challenge in cancer treatment. Secreted protein acidic and rich in cysteine (SPARC) has high binding affinity to albumin and has been shown to enhance the penetration and uptake of albumin-based drug carriers in tumors. Here, we developed a strategy to alter the tumor microenvironment (TME) by upregulating SPARC to enhance the delivery efficiency of albumin-based drug carriers into tumors. We prepared albumin nanoparticles encapsulating an NF-κB controllable CRISPR activation system (SP-NPs). SP-NPs achieved tumor-selective SPARC upregulation by responding to the highly activated NF-κB in tumor cells. Whereas a single dose of SP-NPs only modestly upregulated SPARC expression, serial administration of SP-NPs created a positive feedback loop that induced progressive increases in SPARC expression as well as tumor cell uptake and tumor penetration of the nanoparticles in vitro, in organoids, and in subcutaneous tumors in vivo. Additionally, pre-treatment with SP-NPs significantly enhanced the anti-tumor efficacy of Abraxane, a commercialized albumin-bound paclitaxel nanoformulation. Our data provide evidence that modulating SPARC in the TME can enhance the efficiency of albumin-based drug delivery to solid tumors, which may result in new strategies to increase the efficacy of nanoparticle-based cancer drugs.

Activation of Young LINE-1 Elements by CRISPRa.

Long interspersed element-1 (LINE-1; L1s) are mobile genetic elements that comprise nearly 20% of the human genome. L1s have been shown to have important functions in various biological processes, and their dysfunction is thought to be linked with diseases and cancers. However, the roles of the repetitive elements are largely not understood. While the CRISPR activation (CRISPRa) system based on catalytically deadCas9 (dCas9) is widely used for genome-wide interrogation of gene function and genetic interaction, few studies have been conducted on L1s. Here, we report using the CRISPRa method to efficiently activate L1s in human L02 cells, a derivative of the HeLa cancer cell line. After CRISPRa, the young L1 subfamilies such as L1HS/L1PA1 and L1PA2 are found to be expressed at higher levels than the older L1s. The L1s with high levels of transcription are closer to full-length and are more densely occupied by the YY1 transcription factor. The activated L1s can either be mis-spliced to form chimeric transcripts or act as alternative promoters or enhancers to facilitate the expression of neighboring genes. The method described here can be used for studying the functional roles of young L1s in cultured cells of interest.

Abstract 15721: CRISPR Activates Heterochromatic Cardiac Genes in Fibroblasts to Initiate Lineage Conversion Into Cardiac Progenitors

Introduction: Our previous studies have demonstrated that CRISPR activation (CRISPRa) of endogenous genes was sufficient to convert fibroblasts into induced cardiovascular progenitor cells (iCPCs) for myocardial regenerative therapy. However, reprogramming efficiency remains a challenge and little has been elucidated about the epigenetic mechanisms of cellular reprogramming, which will be important for methodological improvements or refinements. Hypothesis: CRISPRa complex can alter the chromatin accessibility and activity of heterochromatic cardiac genes in fibroblasts, directly initiating cardiac reprogramming. Methods: A fibroblast-specific CRISPRa mice model (Pdgfra-cre/ERT; ROSA-dCas9-SunTag) was generated. SgRNAs targeting the promoter region of Gata4, Isl1, Nkx2-5, Baf60c, or Tbx5 genes were transduced through an AAV system in cardiac fibroblast isolated from transgenic mice. iCPCs were confirmed via immunostaining of CPC markers and cardiac electrophysiological assays. iCPCs were collected for RNA-seq, ATAC-seq, and ChIP-seq after reprogramming induction to reveal the epigenetic landscape modulated by CRISPRa. Results: The CRISPRa system was activated in ~80% of fibroblasts after tamoxifen induction and AAV-sgRNA transfection. The reprogrammed iCPCs were characterized by immunostaining of proliferative markers (Ki67 and Pcna), progenitor surface markers (Flk1 and Ssea1), and markers of cardiac tri-lineage cells after induced differentiation. RNA-seq data showed the upregulation of genes associated with cell cycling (Cdkn1b, Aurkb, etc.) and heart development (Myh6, Irx4, etc.) in CRISPRa-induced cells. ATAC-seq showed increased chromatin accessibility at the loci of cardiogenic genes targeted by CRISPRa in fibroblasts. Furthermore, ChIP-seq of Gata4, Nkx2.5, and Tbx5 associated DNA shows that these factors can bind to their own promoter regions in fibroblasts after CRISPRa induction. Conclusions: CRISPRa complexes open the chromatin of the targeted genes in fibroblasts and establish an endogenous autoregulatory loop to ensure stable reprogramming into cardiac progenitors.

UCHL1 alleviates apoptosis in chondrocytes via upregulation of HIF‑1α‑mediated mitophagy.

Stem cell‑based tissue engineering has shown significant potential for rapid restoration of injured cartilage tissues. Stem cells frequently undergo apoptosis because of the prevalence of oxidative stress and inflammation in the microenvironment at the sites of injury. Our previous study demonstrated that stabilization of hypoxia‑inducible factor1α (HIF‑1α) is key to resisting apoptosis in chondrocytes. Recently, it was reported that Ubiquitin C‑terminal hydrolaseL1 (UCHL1) can stabilize HIF‑1α by abrogating the ubiquitination process. However, the effect of UCHL1 on apoptosis in chondrocytes remains unclear. Herein, adipose‑derived stem cells were differentiated into chondrocytes. Next, the CRISPR activation (CRISPRa) system, LDN‑57444 (LDM; a specific inhibitor for UCHL1), KC7F2 (a specific inhibitor for HIF‑1α), and 3‑methyladenine (a specific inhibitor for mitophagy) were used to activate or block UCHL1, HIF‑1α, and mitophagy. Mitophagy, apoptosis, and mitochondrial function in chondrocytes were detected using immunofluorescence, TUNEL staining, and flow cytometry. Moreover, the oxygen consumption rate of chondrocytes was measured using the Seahorse XF 96 Extracellular Flux Analyzer. UCHL1 expression was increased in hypoxia, which in turn regulated mitophagy and apoptosis in the chondrocytes. Further studies revealed that UCHL1 mediated hypoxia‑regulated mitophagy in the chondrocytes. The CRISPRa module was utilized to activate UCHL1 effectively for 7days; endogenous activation of UCHL1 accelerated mitophagy, inhibited apoptosis, and maintained mitochondrial function in the chondrocytes, which was mediated by HIF‑1α. Taken together, UCHL1 could block apoptosis in chondrocytes via upregulation of HIF‑1α-mediated mitophagy and maintain mitochondrial function. These results indicate the potential of UCHL1 activation using the CRISPRa system for the regeneration of cartilage tissue.

Targeted shock-and-kill HIV-1 gene therapy approach combining CRISPR activation, suicide gene tBid and retargeted adenovirus delivery

Infections with the human immunodeficiency virus type 1 (HIV-1) are incurable due the long-lasting, latent viral reservoir. The shock-and-kill cure approach aims to activate latent proviruses in HIV-1 infected cells and subsequently kill these cells with strategies such as therapeutic vaccines or immune enhancement. Here, we combined the dCas9-VPR CRISPR activation (CRISPRa) system with gRNA-V, the truncated Bid (tBid)-based suicide gene strategy and CD3-retargeted adenovirus (Ad) delivery vectors, in an all-in-one targeted shock-and-kill gene therapy approach to achieve specific elimination of latently HIV-1 infected cells. Simultaneous transduction of latently HIV-1 infected J-Lat 10.6 cells with a CD3-retargeted Ad-CRISPRa-V and Ad-tBid led to a 57.7 ± 17.0% reduction of productively HIV-1 infected cells and 2.4-fold ± 0.25 increase in cell death. The effective activation of latent HIV-1 provirus by Ad-CRISPRa-V was similar to the activation control TNF-α. The strictly HIV-1 dependent and non-leaky killing by tBid could be demonstrated. Furthermore, the high transduction efficiencies of up to 70.8 ± 0.4% by the CD3-retargeting technology in HIV-1 latently infected cell lines was the basis of successful shock-and-kill. This novel targeted shock-and-kill all-in-one gene therapy approach has the potential to safely and effectively eliminate HIV-1 infected cells in a highly HIV-1 and T cell specific manner.

CRISPR/Cas9-based gene activation and base editing in Populus.

The genus Populus has long been used for environmental, agroforestry and industrial applications worldwide. Today Populus is also recognized as a desirable crop for biofuel production and a model tree for physiological and ecological research. As such, various modern biotechnologies, including CRISPR/Cas9-based techniques, have been actively applied to Populus for genetic and genomic improvements for traits such as increased growth rate and tailored lignin composition. However, CRISPR/Cas9 has been primarily used as the active Cas9 form to create knockouts in the hybrid poplar clone "717-1B4" (P. tremula x P. alba clone INRA 717-1B4). Alternative CRISPR/Cas9-based technologies, e.g. those involving modified Cas9 for gene activation and base editing, have not been evaluated in most Populus species for their efficacy. Here we employed a deactivated Cas9 (dCas9)-based CRISPR activation (CRISPRa) technique to fine-tune the expression of two target genes, TPX2 and LecRLK-G which play important roles in plant growth and defense response, in hybrid poplar clone "717-1B4" and poplar clone "WV94" (P. deltoides "WV94"), respectively. We observed that CRISPRa resulted in 1.2-fold to 7.0-fold increase in target gene expression through transient expression in protoplasts and Agrobacterium-mediated stable transformation, demonstrating the effectiveness of dCas9-based CRISPRa system in Populus. In addition, we applied Cas9 nickase (nCas9)-based cytosine base editor (CBE) to precisely introduce premature stop codons via C-to-T conversion, with an efficiency of 13%-14%, in the target gene PLATZ which encodes a transcription factor involved in plant fungal pathogen response in hybrid poplar clone "717-1B4". Overall, we showcase the successful application of CRISPR/Cas-based technologies in gene expression regulation and precise gene engineering in two Populus species, facilitating the adoption of emerging genome editing tools in woody species.

Validation of CRISPR activation system in Aedes cells using multicistronic plasmid vectors.

Aedes mosquitoes transmit several pathogens including flaviviruses to humans which result in high morbidity and mortality. Owing to adaptability and climate change, these mosquito vectors are predicted to establish in new geographical areas thus exposing larger populations to the risk of infection. Therefore, control of Aedes vector is necessary to prevent disease transmission. Recently, genetic approaches to vector control have shown promise; however, the tools and methods for manipulating the mosquito genome are rather limited. While CRISPR-Cas9 system has been adapted for gene editing purposes in Aedes mosquito, the dCas9-based transcription control of genes remain unexplored. In this study we report implementation of the CRISPR activation system in Aedes cells. For this we designed, constructed and tested a bi-partite plasmid-based strategy that allows expression of the dCas9-VPR and targeting guide RNA together with a reporter cassette. Quantitative analysis of the fluorescent reporter gene levels showed a robust over-expression validating CRISPR activation in Aedes cells. This strategy and the biological parts will be useful resource for synthetic transcription factor-based robust upregulation of Aedes genes for application of synthetic biology approaches for vector control.

Split dCas12a activator for lncRNA H19 activation to enhance BMSC differentiation and promote calvarial bone healing

Healing of large calvarial bone defects in adults is challenging. We previously showed that inducing chondrogenic differentiation of mesenchymal stem cells from bone marrow (BMSC) or adipose tissue (ASC) before implantation can switch the repair pathway and improve calvarial bone healing. Split dCas12a activator is a new CRISPR activation system comprising the amino (N) and carboxyl (C) fragments of dCas12a protein, each being fused with synthetic transcription activators at both termini. The split dCas12a activator was shown to induce programmable gene expression in cell lines. Here we exploited the split dCas12a activator to activate the expression of chondroinductive long non-coding RNA H19. We showed that co-expression of the split N- and C-fragments resulted in spontaneous dimerization, which elicited stronger activation of H19 than full-length dCas12a activator in rat BMSC and ASC. We further packaged the entire split dCas12a activator system (13.2 kb) into a hybrid baculovirus vector, which enhanced and prolonged H19 activation for at least 14 days in BMSC and ASC. The extended H19 activation elicited potent chondrogenic differentiation and inhibited adipogenesis. Consequently, the engineered BMSC promoted in vitro cartilage formation and augmented calvarial bone healing in rats. These data implicated the potentials of the split dCas12a activator for stem cell engineering and regenerative medicine.

Transplantation of Adipose-Tissue-Engineered Constructs with CRISPR-Mediated UCP1 Activation.

Thermogenic adipocytes have potential utility for the development of approaches to treat type 2 diabetes and obesity-associated diseases. Although several reports have proved the positive effect of beige and brown adipocyte transplantation in obese mice, translation to human cell therapy needs improvement. Here, we describe the application of CRISPR activation (CRISPRa) technology for generating safe and efficient adipose-tissue-engineered constructs with enhanced mitochondrial uncoupling protein 1 (UCP1) expression. We designed the CRISPRa system for the activation of UCP1 gene expression. CRISPRa-UCP1 was delivered into mature adipocytes by a baculovirus vector. Modified adipocytes were transplanted in C57BL/6 mice, followed by analysis of grafts, inflammation and systemic glucose metabolism. Staining of grafts on day 8 after transplantation shows them to contain UCP1-positive adipocytes. Following transplantation, adipocytes remain in grafts and exhibit expression of PGC1α transcription factor and hormone sensitive lipase (HSL). Transplantation of CRISPRa-UCP1-modified adipocytes does not influence glucose metabolism or inflammation in recipient mice. We show the utility and safety of baculovirus vectors for CRISPRa-based thermogenic gene activation. Our findings suggest a means of improving existing cell therapy approaches using baculovirus vectors and CRISPRa for modification and transplantation of non-immunogenic adipocytes.

CRISPR mediated transactivation in the human disease vector Aedes aegypti.

As a major insect vector of multiple arboviruses, Aedes aegypti poses a significant global health and economic burden. A number of genetic engineering tools have been exploited to understand its biology with the goal of reducing its impact. For example, current tools have focused on knocking-down RNA transcripts, inducing loss-of-function mutations, or expressing exogenous DNA. However, methods for transactivating endogenous genes have not been developed. To fill this void, here we developed a CRISPR activation (CRISPRa) system in Ae. aegypti to transactivate target gene expression. Gene expression is activated through pairing a catalytically-inactive ('dead') Cas9 (dCas9) with a highly-active tripartite activator, VP64-p65-Rta (VPR) and synthetic guide RNA (sgRNA) complementary to a user defined target-gene promoter region. As a proof of concept, we demonstrate that engineered Ae. aegypti mosquitoes harboring a binary CRISPRa system can be used to effectively overexpress two developmental genes, even-skipped (eve) and hedgehog (hh), resulting in observable morphological phenotypes. We also used this system to overexpress the positive transcriptional regulator of the Toll immune pathway known as AaRel1, which resulted in a significant suppression of dengue virus serotype 2 (DENV2) titers in the mosquito. This system provides a versatile tool for research pathways not previously possible in Ae. aegypti, such as programmed overexpression of endogenous genes, and may aid in gene characterization studies and the development of innovative vector control tools.

Targeted Activation of Arabidopsis Genes by a Potent CRISPR-Act3.0 System.

The CRISPR/Cas system has emerged as a versatile platform for sequence-specific genome engineering in plants. Beyond genome editing, CRISPR/Cas systems, based on nuclease-deficient Cas9 (dCas9), have been repurposed as an RNA-guided platform for transcriptional regulation. CRISPR activation (CRISPRa) represents a novel gain-of-function (GOF) strategy, conferring robust over-expression of the target gene within its native chromosomal context. The CRISPRa systems enable precise, scalable, and robust RNA-guided transcription activation, holding great potential for a variety of fundamental and translational research. In this chapter, we provide a step-by-step guide for efficient gene activation in Arabidopsis based on a highly robust CRISPRa system, CRISPR-Act3.0. We present detailed procedures onthe sgRNA design, CRISPR-Act3.0 system construction, Agrobacterium-mediated transformation of Arabidopsis using the floral dip method, and identification of desired transgenic plants.

Endosomal Escapable and Nuclear Localizing Cationic Polyaspartate-Based CRISPR Activation System for Preventing Respiratory Virus Infection by Specifically Inducing Interferon-λ.

Global pandemics caused by viruses cause widespread panic and economic losses. The lack of specific antivirals and vaccines increases the spreading of viral diseases worldwide. Thus, alternative strategies are required to manage viral outbreaks. Here, we develop a CRISPR activation (CRISPRa) system based on polymeric carriers to prevent respiratory virus infection in a mouse model. A polyaspartate grafted with 2-(diisopropylamino) ethylamine (DIP) and nuclear localization signal peptides (NLS-MTAS fusion peptide) was complexed with plasmid DNA (pDNA) encoding dCas9-VPR and sgRNA targeting IFN-λ. The pH-sensitive DIP and NLS-MTAS groups were favor of endo-lysosomal escape and nuclear localization of pDNA, respectively. They synergistically improved gene transfection efficiency, resulting in significant reporter gene expression and IFN-λ upregulation in lung tissue. In vitro and in vivo prophylactic experiments showed that the non-viral CRISPRa system could prevent infection caused by H1N1 viruses with minimal inflammatory responses, presenting a promising prophylactic approach against respiratory virus infections.

Cross‐Platform Synaptic Network Analysis of Alzheimer’s Disease Contextualizes Proteomic Targets for Functional Validation

AbstractBackgroundCognitive decline in Alzheimer’s disease (AD) correlates well with the extent of tau pathology and synapse or dendritic spine loss. Thus, studying synaptic processes may aid in the identification of new therapeutic targets that limit cognitive decline. The entorhinal cortex (EC) is an initial site of tau tangle formation, from which tau can propagate to other brain regions through synaptic connections. We developed a pipeline whereby dendritic spine density and morphology as well as synaptic tau measurements from the EC were incorporated into a synaptic proteomic network to identify mechanisms of synaptic pathology in AD patients.MethodGolgi‐stained dendrites from postmortem human BA28 EC were imaged using high‐resolution brightfield microscopy and digitally reconstructed in 3D for morphometric analysis. Synaptosome fractions were isolated from the same BA28 samples. Levels of synaptosome and insoluble phosphorylated tau were measured by ELISA. Additionally, liquid chromatography coupled with tandem mass spectrometry‐based proteomics was performed on synaptosomes. Weighted Gene Co‐Expression Network Analysis (WGCNA) was used to generate a synaptic network of protein co‐expression modules. Module expression was correlated with dendritic spine measurements and phospho‐tau levels to guide selection of proteins for validation in model systems. Validation experiments were performed in neurons and Neuro‐2a cells, using a CRISPR activation system for target gene upregulation.ResultSpine density in the EC is reduced in AD, but not in cognitively normal individuals with AD pathology (CAD) cases. AD cases exhibit more synaptosomal pS396 tau than CAD cases. Module expression was correlated with dendritic spine density and morphology. We identified TWF2 as a hub protein of Module 16, which is positively correlated with thin spine length. Twf2 upregulation increased thin spine length in cultured neurons. Module expression was then correlated with phospho‐tau levels. We identified PPP1R7 as a hub protein of Module 24, which is inversely correlated with synaptosomal pS396 tau in human EC.ConclusionWe demonstrate that incorporating orthogonal cellular and molecular measurements into a proteomic network enables unbiased identification of proteins functionally involved in regulating those traits. Beyond elucidating synaptic processes in AD, this pipeline offers a blueprint to better contextualize omics‐based targets with relevant biological mechanisms.