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Equitable and accessible education in life sciences, bioengineering, and synthetic biology is crucial for training the next generation of scientists, fostering transparency in public decision-making, and ensuring biotechnology can benefit a wide-ranging population. As a groundbreaking technology for genome engineering, CRISPR has transformed research and therapeutics. However, hands-on exposure to this technology in educational settings remains limited due to the extensive resources required for CRISPR experiments. Here, we develop CRISPRkit, an affordable kit designed for gene editing and regulation in high school education. CRISPRkit eliminates the need for specialized equipment, prioritizes biosafety, and utilizes cost-effective reagents. By integrating CRISPRi gene regulation, colorful chromoproteins, cell-free transcription-translation systems, smartphone-based quantification, and an in-house automated algorithm (CRISPectra), our kit offers an inexpensive (~$2) and user-friendly approach to performing and analyzing CRISPR experiments, without the need for a traditional laboratory setup. Experiments conducted by high school students in classroom settings highlight the kit's utility for reliable CRISPRkit experiments. Furthermore, CRISPRkit provides a modular and expandable platform for genome engineering, and we demonstrate its applications for controlling fluorescent proteins and metabolic pathways such as melanin production. We envision CRISPRkit will facilitate biotechnology education for communities of diverse socioeconomic and geographic backgrounds.
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Sistemas CRISPR-Cas , Edição de Genes , Biologia Sintética , Edição de Genes/métodos , Biologia Sintética/métodos , Humanos , Estudantes , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Instituições AcadêmicasRESUMO
The epigenome is a complex framework through which gene expression is precisely and flexibly modulated to incorporate heritable memory and responses to environmental stimuli. It governs diverse cellular processes, including cell fate, disease, and aging. The need to understand this system and precisely control gene expression outputs for therapeutic purposes has precipitated the development of a diverse set of epigenetic editing tools. Here, we review the existing toolbox for targeted epigenetic editing, technical considerations of the current technologies, and opportunities for future development. We describe applications of therapeutic epigenetic editing and their potential for treating disease, with a discussion of ongoing delivery challenges that impede certain clinical interventions, particularly in the brain. With simultaneous advancements in available engineering tools and appropriate delivery technologies, we predict that epigenetic editing will increasingly cement itself as a powerful approach for safely treating a wide range of disorders in all tissues of the body.
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The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelles, the sites of replication of viral genomic RNA (vgRNA). To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain numerous vgRNA molecules along with the replication enzymes and clusters of viral double-stranded RNA (dsRNA). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of endoplasmic reticulum (ER) markers and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are encapsulated into DMVs, which have membranes derived from the host ER. These organelles merge into larger vesicle packets as infection advances. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.
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Retículo Endoplasmático , Organelas , RNA Viral , SARS-CoV-2 , Replicação Viral , SARS-CoV-2/fisiologia , SARS-CoV-2/ultraestrutura , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , RNA Viral/metabolismo , RNA Viral/genética , Replicação Viral/fisiologia , Humanos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Retículo Endoplasmático/ultraestrutura , Organelas/virologia , Organelas/metabolismo , Organelas/ultraestrutura , Chlorocebus aethiops , Células Vero , Animais , COVID-19/virologia , COVID-19/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais/genética , Microscopia de Fluorescência , Compartimentos de Replicação Viral/metabolismo , RNA de Cadeia Dupla/metabolismoRESUMO
Embryonic stem cells (ESCs) can self-organize in vitro into developmental patterns with spatial organization and molecular similarity to that of early embryonic stages. This self-organization of ESCs requires transmission of signaling cues, via addition of small molecule chemicals or recombinant proteins, to induce distinct embryonic cellular fates and subsequent assembly into structures that can mimic aspects of early embryonic development. During natural embryonic development, different embryonic cell types co-develop together, where each cell type expresses specific fate-inducing transcription factors through activation of non-coding regulatory elements and interactions with neighboring cells. However, previous studies have not fully explored the possibility of engineering endogenous regulatory elements to shape self-organization of ESCs into spatially-ordered embryo models. Here, we hypothesized that cell-intrinsic activation of a minimum number of such endogenous regulatory elements is sufficient to self-organize ESCs into early embryonic models. Our results show that CRISPR-based activation (CRISPRa) of only two endogenous regulatory elements in the genome of pluripotent stem cells is sufficient to generate embryonic patterns that show spatial and molecular resemblance to that of pre-gastrulation mouse embryonic development. Quantitative single-cell live fluorescent imaging showed that the emergence of spatially-ordered embryonic patterns happens through the intrinsic induction of cell fate that leads to an orchestrated collective cellular motion. Based on these results, we propose a straightforward approach to efficiently form 3D embryo models through intrinsic CRISPRa-based epigenome editing and independent of external signaling cues. CRISPRa-Programmed Embryo Models (CPEMs) show highly consistent composition of major embryonic cell types that are spatially-organized, with nearly 80% of the structures forming an embryonic cavity. Single cell transcriptomics confirmed the presence of main embryonic cell types in CPEMs with transcriptional similarity to pre-gastrulation mouse embryos and revealed novel signaling communication links between different embryonic cell types. Our findings offer a programmable embryo model and demonstrate that minimum intrinsic epigenome editing is sufficient to self-organize ESCs into highly consistent pre-gastrulation embryo models.
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CRISPR technologies have begun to revolutionize T cell therapies; however, conventional CRISPR-Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed multiplexed effector guide arrays (MEGA), a platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR-Cas13d. MEGA enables quantitative, reversible, and massively multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of T cell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathways to enhance CAR T cell fitness and anti-tumor activity in vitro and in vivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond.
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Engenharia Metabólica , Linfócitos T , Humanos , Perfilação da Expressão Gênica , Engenharia Metabólica/métodos , RNA , TranscriptomaRESUMO
The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelle where the replication of viral genomic RNA (vgRNA) occurs. To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain vgRNA clusters along with viral double-stranded RNA (dsRNA) clusters and the replication enzyme, encapsulated by membranes derived from the host endoplasmic reticulum (ER). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of ER labels and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are enclosed by DMVs at early infection stages which then merge into vesicle packets as infection progresses. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.
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Manipulating gene expression in the host genome with high precision is crucial for controlling cellular function and behavior. Here, we present a precise, non-invasive, and tunable strategy for controlling the expression of multiple endogenous genes both in vitro and in vivo, utilizing ultrasound as the stimulus. By engineering a hyper-efficient dCas12a and effector under a heat shock promoter, we demonstrate a system that can be inducibly activated through thermal energy produced by ultrasound absorption. This system allows versatile thermal induction of gene activation or base editing across cell types, including primary T cells, and enables multiplexed gene activation using a single guide RNA array. In mouse models, localized temperature elevation guided by high-intensity focused ultrasound effectively triggers reporter gene expression in implanted cells. Our work underscores the potential of ultrasound as a clinically viable approach to enhance cell and gene-based therapies via precision genome and epigenome engineering.
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Edição de Genes , Genoma , Animais , Camundongos , Genoma/genética , Terapia Genética , Epigenoma , Genes Reporter , Sistemas CRISPR-Cas/genéticaRESUMO
The pleiotropic benefits of statins in cardiovascular diseases that are independent of their lipid-lowering effects have been well documented, but the underlying mechanisms remain elusive. Here we show that simvastatin significantly improves human induced pluripotent stem cell-derived endothelial cell functions in both baseline and diabetic conditions by reducing chromatin accessibility at transcriptional enhanced associate domain elements and ultimately at endothelial-to-mesenchymal transition (EndMT)-regulating genes in a yes-associated protein (YAP)-dependent manner. Inhibition of geranylgeranyltransferase (GGTase) I, a mevalonate pathway intermediate, repressed YAP nuclear translocation and YAP activity via RhoA signaling antagonism. We further identified a previously undescribed SOX9 enhancer downstream of statin-YAP signaling that promotes the EndMT process. Thus, inhibition of any component of the GGTase-RhoA-YAP-SRY box transcription factor 9 (SOX9) signaling axis was shown to rescue EndMT-associated endothelial dysfunction both in vitro and in vivo, especially under diabetic conditions. Overall, our study reveals an epigenetic modulatory role for simvastatin in repressing EndMT to confer protection against endothelial dysfunction.
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Successful implementation of adoptive cell therapy (ACT) of cancer requires comprehensively addressing biological and practical challenges. This approach has been largely overlooked, resulting in a gap between the potential of ACT and its actual effectiveness. We summarize the most promising technical strategies in creating an "ideal" ACT product, focusing on chimeric antigen receptor (CAR)-engineered cells. Since many requirements for effective ACT are common to most cancers, what we outline here might have a broader impact.
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Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia Adotiva , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
Chronic wounds impose a significant healthcare burden to a broad patient population. Cell-based therapies, while having shown benefits for the treatment of chronic wounds, have not yet achieved widespread adoption into clinical practice. We developed a CRISPR/Cas9 approach to precisely edit murine dendritic cells to enhance their therapeutic potential for healing chronic wounds. Using single-cell RNA sequencing of tolerogenic dendritic cells, we identified N-myc downregulated gene 2 (Ndrg2), which marks a specific population of dendritic cell progenitors, as a promising target for CRISPR knockout. Ndrg2-knockout alters the transcriptomic profile of dendritic cells and preserves an immature cell state with a strong pro-angiogenic and regenerative capacity. We then incorporated our CRISPR-based cell engineering within a therapeutic hydrogel for in vivo cell delivery and developed an effective translational approach for dendritic cell-based immunotherapy that accelerated healing of full-thickness wounds in both non-diabetic and diabetic mouse models. These findings could open the door to future clinical trials using safe gene editing in dendritic cells for treating various types of chronic wounds.
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Sistemas CRISPR-Cas , Traumatismos Craniocerebrais , Humanos , Camundongos , Animais , Cicatrização/genética , Genes myc , Edição de Genes , Células DendríticasRESUMO
The targeted insertion and stable expression of a large genetic payload in primary human cells demands methods that are robust, efficient and easy to implement. Large payload insertion via retroviruses is typically semi-random and hindered by transgene silencing. Leveraging homology-directed repair to place payloads under the control of endogenous essential genes can overcome silencing but often results in low knock-in efficiencies and cytotoxicity. Here we report a method for the knock-in and stable expression of a large payload and for the simultaneous knock-in of two genes at two endogenous loci. The method, which we named CLIP (for 'CRISPR for long-fragment integration via pseudovirus'), leverages an integrase-deficient lentivirus encoding a payload flanked by homology arms and 'cut sites' to insert the payload upstream and in-frame of an endogenous essential gene, followed by the delivery of a CRISPR-associated ribonucleoprotein complex via electroporation. We show that CLIP enables the efficient insertion and stable expression of large payloads and of two difficult-to-express viral antigens in primary T cells at low cytotoxicity. CLIP offers a scalable and efficient method for manufacturing engineered primary cells.
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Integrases , Lentivirus , Humanos , Lentivirus/genética , Integrases/genética , Integrases/metabolismo , Técnicas de Introdução de Genes , Transgenes/genética , Reparo de DNA por RecombinaçãoRESUMO
BACKGROUND: Chimeric antigen receptor (CAR)-T cell therapies for the treatment of hematological malignancies experienced tremendous progress in the last decade. However, essential limitations need to be addressed to further improve efficacy and reduce toxicity to assure CAR-T cell persistence, trafficking to the tumor site, resistance to an hostile tumor microenvironment (TME), and containment of toxicity restricting production of powerful but potentially toxic bioproducts to the TME; the last could be achieved through contextual release upon tumor antigen encounter of factors capable of converting an immune suppressive TME into one conducive to immune rejection. METHODS: We created an HER2-targeting CAR-T (RB-312) using a clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system, which induces the expression of the IL-12 heterodimer via conditional transcription of its two endogenous subunits p35 and p40. This circuit includes two lentiviral constructs. The first one (HER2-TEV) expresses an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3z co-stimulatory domains linked to the tobacco etch virus (TEV) protease and two single guide RNAs (sgRNA) targeting the interleukin (IL)-12A and IL12B transcription start site (TSS), respectively. The second construct (LdCV) encodes linker for activation of T cells (LAT) fused to nuclease-deactivated Streptococcus Pyogenes Cas9 (dCas9)-VP64-p65-Rta (VPR) via a TEV-cleavable sequence (TCS). Activation of the CAR brings HER2-TEV in close proximity to LdCV releasing dCas9 for nuclear localization. This conditional circuit leads to conditional and reversible induction of the IL-12/p70 heterodimer. RB-312 was compared in vitro to controls (cRB-312), lacking the IL-12 sgRNAs and conventional HER2 CAR (convCAR). RESULTS: The inducible CRISPRa system activated endogenous IL-12 expression resulting in enhanced secondary interferon (FN)-γ production, cytotoxicity, and CAR-T proliferation in vitro, prolonged in vivo persistence and greater suppression of HER2+ FaDu oropharyngeal cancer cell growth compared to the conventional CAR-T cell product. No systemic IL-12 was detected in the peripheral circulation. Moreover, the combination with programmed death ligand (PD-L1) blockade demonstrated robust synergistic effects. CONCLUSIONS: RB-312, the first clinically relevant product incorporating a CRISPRa system with non-gene editing and reversible upregulation of endogenous gene expression that promotes CAR-T cells persistence and effectiveness against HER2-expressing tumors. The autocrine effects of reversible, nanoscale IL-12 production limits the risk of off-tumor leakage and systemic toxicity.
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Imunoterapia Adotiva , Neoplasias , Receptores de Antígenos Quiméricos , Antígeno B7-H1 , Antígenos CD28 , Interleucina-12/genética , Ligantes , Neoplasias/terapia , Sistemas de Liberação de MedicamentosRESUMO
The Central Dogma of the flow of genetic information is arguably the crowning achievement of 20th century molecular biology. Reversing the flow of information from RNA to DNA or chromatin has come to the fore in recent years, from the convergence of fundamental discoveries and synthetic biology. Inspired by the example of long noncoding RNAs (lncRNAs) in mammalian genomes that direct chromatin modifications and gene expression, synthetic biologists have repurposed prokaryotic RNA-guided genome defense systems such as CRISPR to edit eukaryotic genomes and epigenomes. Here we explore the parallels of these two fields and highlight opportunities for synergy and future breakthroughs.
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Edição de Genes , RNA Longo não Codificante , Animais , DNA/genética , Epigênese Genética , Cromatina/genética , RNA Longo não Codificante/genética , Sistemas CRISPR-Cas/genética , Mamíferos/metabolismoRESUMO
Designer T cells offer a novel paradigm for treating diseases like cancer, yet they are often hindered by target recognition evasion and limited in vivo control. To overcome these challenges, we develop valency-controlled receptors (VCRs), a novel class of synthetic receptors engineered to enable precise modulation of immune cell activity. VCRs use custom-designed valency-control ligands (VCLs) to modulate T cell signaling via spatial molecular clustering. Using multivalent DNA origami as VCL, we first establish that valency is important for tuning the activity of CD3-mediated immune activation. We then generate multivalent formats of clinically relevant drugs as VCL and incorporate VCR into the architecture of chimeric antigen receptors (CARs). Our data demonstrate that VCL-mediated VCRs can significantly amplify CAR activities and improve suboptimal CARs. Finally, through medicinal chemistry, we synthesize programmable, bioavailable VCL drugs that potentiate targeted immune response against low-antigen tumors both in vitro and in vivo. Our findings establish receptor valency as a core mechanism for enhancing CAR functionality and offer a synthetic chemical biology platform for strengthening customizable, potent, and safer cell therapies.
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The clustered regularly interspaced short palindromic repeats (CRISPR) renaissance was catalysed by the discovery that RNA-guided prokaryotic CRISPR-associated (Cas) proteins can create targeted double-strand breaks in mammalian genomes. This finding led to the development of CRISPR systems that harness natural DNA repair mechanisms to repair deficient genes more easily and precisely than ever before. CRISPR has been used to knock out harmful mutant genes and to fix errors in coding sequences to rescue disease phenotypes in preclinical studies and in several clinical trials. However, most genetic disorders result from combinations of mutations, deletions and duplications in the coding and non-coding regions of the genome and therefore require sophisticated genome engineering strategies beyond simple gene knockout. To overcome this limitation, the toolbox of natural and engineered CRISPR-Cas systems has been dramatically expanded to include diverse tools that function in human cells for precise genome editing and epigenome engineering. The application of CRISPR technology to edit the non-coding genome, modulate gene regulation, make precise genetic changes and target infectious diseases has the potential to lead to curative therapies for many previously untreatable diseases.
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Sistemas CRISPR-Cas , Edição de Genes , Animais , Humanos , Genoma , Reparo do DNA , Mamíferos/genéticaRESUMO
Natural killer (NK) cells are cytotoxic effector cells that target and lyse virally infected cells; many viruses therefore encode mechanisms to escape such NK cell killing. Here, we interrogate the ability of SARS-CoV-2 to modulate NK cell recognition and lysis of infected cells. We find that NK cells exhibit poor cytotoxic responses against SARS-CoV-2-infected targets, preferentially killing uninfected bystander cells. We demonstrate that this escape is driven by downregulation of ligands for the activating receptor NKG2D (NKG2D-L). Indeed, early in viral infection, prior to NKG2D-L downregulation, NK cells are able to target and kill infected cells; however, this ability is lost as viral proteins are expressed. Finally, we find that SARS-CoV-2 non-structural protein 1 (Nsp1) mediates downregulation of NKG2D-L and that Nsp1 alone is sufficient to confer resistance to NK cell killing. Collectively, our work demonstrates that SARS-CoV-2 evades direct NK cell cytotoxicity and describes a mechanism by which this occurs.
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COVID-19 , Subfamília K de Receptores Semelhantes a Lectina de Células NK , SARS-CoV-2 , Proteínas não Estruturais Virais , Humanos , Morte Celular , COVID-19/metabolismo , Regulação para Baixo , Células Matadoras Naturais/metabolismo , Ligantes , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , SARS-CoV-2/metabolismoRESUMO
Mammalian genomes have multiple enhancers spanning an ultralong distance (>megabases) to modulate important genes, but it is unclear how these enhancers coordinate to achieve this task. We combine multiplexed CRISPRi screening with machine learning to define quantitative enhancer-enhancer interactions. We find that the ultralong distance enhancer network has a nested multilayer architecture that confers functional robustness of gene expression. Experimental characterization reveals that enhancer epistasis is maintained by three-dimensional chromosomal interactions and BRD4 condensation. Machine learning prediction of synergistic enhancers provides an effective strategy to identify noncoding variant pairs associated with pathogenic genes in diseases beyond genome-wide association studies analysis. Our work unveils nested epistasis enhancer networks, which can better explain enhancer functions within cells and in diseases.