CRISPR Activation System Research Articles

Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans.

For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans, which employs a guide RNA to target an activator complex to the promoter region of a gene of interest, thus driving transcriptional expression of that gene. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust and constitutive overexpression. We further demonstrate the specificity of the system via RNA sequencing. We highlight the application of CRISPR activation to overexpress genes involved in pathogenesis and drug susceptibility, and contribute toward the identification of novel phenotypes. Consequently, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.

Identification of NRAS Downstream Genes with CRISPR Activation Screening.

Mutations in NRAS constitutively activate cell proliferation signaling in malignant neoplasms, such as leukemia and melanoma, and the clarification of comprehensive downstream genes of NRAS might lead to the control of cell-proliferative signals of NRAS-driven cancers. We previously established that NRAS expression and proliferative activity can be controlled with doxycycline and named as THP-1 B11. Using a CRISPR activation library on THP-1 B11 cells with the NRAS-off state, survival clones were harvested, and 21 candidate genes were identified. By inducting each candidate guide RNA with the CRISPR activation system, DOHH, HIST1H2AC, KRT32, and TAF6 showed higher cell-proliferative activity. The expression of DOHH, HIST1H2AC, and TAF6 was definitely upregulated with NRAS expression. Furthermore, MEK inhibitors resulted in the decreased expression of DOHH, HIST1H2AC, and TAF6 proteins in parental THP-1 cells. The knockdown of DOHH, HIST1H2AC, and TAF6 was found to reduce proliferation in THP-1 cells, indicating that they are involved in the downstream proliferation of NRAS. These molecules are expected to be new therapeutic targets for NRAS-mutant leukemia cells.

Non-viral CRISPR activation system targeting VEGF-A and TGF-β1 for enhanced osteogenesis of pre-osteoblasts implanted with dual-crosslinked hydrogel.

Healing of large calvarial bone defects remains challenge but may be improved by stimulating bone regeneration of implanted cells. The aim of this study is to specially co-activate transforming growth factor β1 (TGF-β1) and vascular endothelial growth factor (VEGF-A) genes expressions in pre-osteoblast MC3T3-E1 cells through the non-viral CRISPR activation (CRISPRa) system to promote osteogenesis. A cationic copolymer carrying nucleus localizing peptides and proton sponge groups dimethyl-histidine was synthesized to deliver CRISPRa system into MC3T3-E1 cells with high cellular uptake, lysosomal escape, and nuclear translocation, which activated VEGF-A and TGF-β1 genes expressions and thereby additively or synergistically induced several osteogenic genes expressions. A tunable dual-crosslinked hydrogel was developed to implant the above engineered cells into mice calvaria bone defect site to promote bone healing in vivo. The combination of multi-genes activation through non-viral CRISPRa system and tunable dual-crosslinked hydrogel provides a versatile strategy for promoting bone healing with synergistic effect.

CRISPR activation screen identifies TGFβ-associated PEG10 as a crucial tumor suppressor in Ewing sarcoma

As the second most common pediatric bone and soft tissue tumor, Ewing sarcoma (ES) is an aggressive disease with a pathognomonic chromosomal translocation t(11;22) resulting in expression of EWS-FLI1, an “undruggable” fusion protein acting as transcriptional modulator. EWS-FLI1 rewires the protein expression in cancer cells by activating and repressing a multitude of genes. The role and contribution of most repressed genes remains unknown to date. To address this, we established a CRISPR activation system in clonal SKNMC cell lines and interrogated a custom focused library covering 871 genes repressed by EWS-FLI1. Among the hits several members of the TGFβ pathway were identified, where PEG10 emerged as prime candidate due to its strong antiproliferative effect. Mechanistic investigations revealed that PEG10 overexpression caused cellular dropout via induction of cell death. Furthermore, non-canonical TGFβ pathways such as RAF/MEK/ERK, MKK/JNK, MKK/P38, known to lead to apoptosis or autophagy, were highly activated upon PEG10 overexpression. Our study sheds new light onto the contribution of TGFβ signalling pathway repression to ES tumorigenesis and suggest that its re-activation might constitute a novel therapeutic strategy.

Inducible CRISPR activation screen for interferon-stimulated genes identifies OAS1 as a SARS-CoV-2 restriction factor.

Interferons establish an antiviral state through the induction of hundreds of interferon-stimulated genes (ISGs). The mechanisms and viral specificities for most ISGs remain incompletely understood. To enable high-throughput interrogation of ISG antiviral functions in pooled genetic screens while mitigating potentially confounding effects of endogenous interferon and antiproliferative/proapoptotic ISG activities, we adapted a CRISPR-activation (CRISPRa) system for inducible ISG expression in isogenic cell lines with and without the capacity to respond to interferons. We used this platform to screen for ISGs that restrict SARS-CoV-2. Results included ISGs previously described to restrict SARS-CoV-2 and novel candidate antiviral factors. We validated a subset of these by complementary CRISPRa and cDNA expression experiments. OAS1, a top-ranked hit across multiple screens, exhibited strong antiviral effects against SARS-CoV-2, which required OAS1 catalytic activity. These studies demonstrate a high-throughput approach to assess antiviral functions within the ISG repertoire, exemplified by identification of multiple SARS-CoV-2 restriction factors.

CRISPR-Mediated Activation of αV Integrin Subtypes Promotes Neuronal Differentiation of Neuroblastoma Neuro2a Cells.

Neuronal differentiation is a complex process whose dysfunction can lead to brain disorders. The development of new tools to target specific steps in the neuronal differentiation process is of paramount importance for a better understanding of the molecular mechanisms involved, and ultimately for developing effective therapeutic strategies for neurodevelopmental disorders. Through their interactions with extracellular matrix proteins, the cell adhesion molecules of the integrin family play essential roles in the formation of functional neuronal circuits by regulating cell migration, neurite outgrowth, dendritic spine formation and synaptic plasticity. However, how different integrin receptors contribute to the successive phases of neuronal differentiation remains to be elucidated. Here, we implemented a CRISPR activation system to enhance the endogenous expression of specific integrin subunits in an in vitro model of neuronal differentiation, the murine neuroblastoma Neuro2a cell line. By combining CRISPR activation with morphological and RT-qPCR analyses, we show that integrins of the αV family are powerful inducers of neuronal differentiation. Further, we identify a subtype-specific role for αV integrins in controlling neurite outgrowth. While αVβ3 integrin initiates neuronal differentiation of Neuro2a cells under proliferative conditions, αVβ5 integrin appears responsible for promoting a complex arborization in cells already committed to differentiation. Interestingly, primary neurons exhibit a complementary expression pattern for β3 and β5 integrin subunits during development. Our findings reveal the existence of a developmental switch between αV integrin subtypes during differentiation and suggest that a timely controlled modulation of the expression of αV integrins by CRISPRa provides a means to promote neuronal differentiation.

Sequential enhancer state remodelling defines human germline competence and specification.

Germline-soma segregation is a fundamental event during mammalian embryonic development. Here, we establish the epigenetic principles of human primordial germ cell (hPGC) development using in vivo hPGCs and stem cell models recapitulating gastrulation. We show that morphogen-induced remodelling of mesendoderm enhancers transiently confers the competence for hPGC fate, but further activation favours mesoderm and endoderm fates. Consistently, reducing the expression of the mesendodermal transcription factor (TF) OTX2 promotes the PGC fate. In hPGCs, SOX17 and TFAP2C initiate activation of enhancers to establish a core germline program, including the transcriptional repressor PRDM1 and pluripotency factors POU5F1 and NANOG. We demonstrate that SOX17 enhancers are the critical components in the regulatory circuitry of germline competence. Furthermore, activation of upstream cis-regulatory elements by an optimised CRISPR activation system is sufficient for hPGC specification. We reveal an enhancer-linked germline TF network that provides the basis for the evolutionary divergence of mammalian germlines.

Uncovering the Distinct Properties of a Bacterial Type I-E CRISPR Activation System.

Synthetic gene regulators based upon CRISPR-Cas systems offer programmable technologies to control gene expression in bacteria. Bacterial CRISPR activators (CRISPRa) have been developed that use engineered type II CRISPR-dCas9 to localize transcription activation domains near promoter elements. However, several reports have demonstrated distance-dependent requirements and periodical activation patterns that overall limit their flexibility. Here, we demonstrate the potential of using an alternative type I-E CRISPR-Cas system to create a CRISPRa with distinct and expanded regulatory properties. Furthermore, we create the first bacterial CRISPRa system based upon a type I-E CRISPR-Cas and characterize the distance-dependent activation patterns to reveal a distinct and more frequent periodicity of activation.

Highly efficient CRISPR systems for loss-of-function and gain-of-function research in pear calli.

CRISPR/Cas systems have been widely used for genome engineering in many plant species. However, their potentials have remained largely untapped in fruit crops, particularly in pear, due to the high levels of genomic heterozygosity and difficulties in tissue culture and stable transformation. To date, only a few reports on the application of the CRISPR/Cas9 system in pear have been documented, and have shown very low editing efficiency. Here we report a highly efficient CRISPR toolbox for loss-of-function and gain-of-function research in pear. We compared four different CRISPR/Cas9 expression systems for loss-of-function analysis and identified a potent system that showed nearly 100% editing efficiency for multi-site mutagenesis. To expand the targeting scope, we further tested different CRISPR/Cas12a and Cas12b systems in pear for the first time, albeit with low editing efficiency. In addition, we established a CRISPR activation (CRISPRa) system for multiplexed gene activation in pear calli for gain-of-function analysis. Furthermore, we successfully engineered the anthocyanin and lignin biosynthesis pathways using both CRISPR/Cas9 and CRISPRa systems in pear calli. Taking these results together, we have built a highly efficient CRISPR toolbox for genome editing and gene regulation, paving the way for functional genomics studies as well as molecular breeding in pear.

Design, Characterization, and Application of Targeted Gene Activation in Bacteria Using a Modular CRISPRa System.

CRISPR-Cas regulators have provided an excellent toolbox to control gene expression due to the versatility of its components and the easy programming of the single guide RNA (sgRNA) to target DNA sequences. Included in this are CRISPR activation (CRISPRa) systems. These systems allow users to activate transcription of a target gene through the localization of transcription activation domains (ADs) near promoter elements, which in turn recruit RNA polymerase (RNAP) to turn on transcription. A variety of different CRISPRa systems have been described that vary in AD type, recruitment strategies, and CRISPR-Cas systems. Recently, a highly modular CRISPRa system was described that allows for facile exchange of ADs and CRISPR-Cas components. This allows for the creation of CRISPRa systems with unique properties, for example, ability to activate from specific positions upstream of a gene of interest. Here, we describe a protocol for designing, characterizing, and applying the modular CRISPRa system for gene activation in E. coli. We first focus on how to identify activating sites upstream of promoters and the cloning of the targeting sgRNA. We then describe how to perform a fluorescence experiment to evaluate activation of a single target site. Finally, we explain how to adapt the system to expand the target range and how to characterize the activation pattern obtained from different CRISPRa designs.

3003 – TARGETED CRISPR-MEDIATED GENE ACTIVATION DURING IN VITRO IPSCS DIFFERENTIATION SUCCESSFULLY EXPANDS ARTERIAL-FATED CELLS IN ASSOCIATION WITH HIGHER PROGENITOR ACTIVITY.

Hematopoietic stem and progenitor cells develop from hemogenic endothelium located in various sites during development. Particularly, Hematopoietic Stem Cells (HSCs) develop from specialised endothelial cells within the wall of the dorsal aorta. This process has proven especially challenging to recapitulate in vitro from pluripotent stem cells. Here, we compared iPSC-derived (PSD) endothelial cells and in vivo (IV) hemogenic endothelium and identified 9 transcription factors expressed at significantly lower levels in vitro. We then developed a novel DOX-inducible CRISPR activation system to induce the expression of those genes during in vitro differentiation. We then employed single-cell RNA sequencing to study the phenotypical changes induced by the target genes. Furthermore, by engineering the gRNA we were able to detect them within the sequencing pipeline. Our data showed a significant expansion of arterial-fated endothelial cells with associated higher in vitro progenitor activity. Interestingly, the expanded arterial cluster is distinct from that expressing hemogenic markers, suggesting that the arterial cells might play a supportive role in the emergence of progenitor cells from the hemogenic endothelium. Hematopoietic stem and progenitor cells develop from hemogenic endothelium located in various sites during development. Particularly, Hematopoietic Stem Cells (HSCs) develop from specialised endothelial cells within the wall of the dorsal aorta. This process has proven especially challenging to recapitulate in vitro from pluripotent stem cells. Here, we compared iPSC-derived (PSD) endothelial cells and in vivo (IV) hemogenic endothelium and identified 9 transcription factors expressed at significantly lower levels in vitro. We then developed a novel DOX-inducible CRISPR activation system to induce the expression of those genes during in vitro differentiation. We then employed single-cell RNA sequencing to study the phenotypical changes induced by the target genes. Furthermore, by engineering the gRNA we were able to detect them within the sequencing pipeline. Our data showed a significant expansion of arterial-fated endothelial cells with associated higher in vitro progenitor activity. Interestingly, the expanded arterial cluster is distinct from that expressing hemogenic markers, suggesting that the arterial cells might play a supportive role in the emergence of progenitor cells from the hemogenic endothelium.

CRISPR activation of endogenous genes reprograms fibroblasts into cardiovascular progenitor cells for myocardial infarction therapy

Fibroblasts can be reprogrammed into cardiovascular progenitor cells (CPCs) using transgenic approaches, although the underlying mechanism remains unclear. We determined whether activation of endogenous genes such as Gata4, Nkx2.5, and Tbx5 can rapidly establish autoregulatory loops and initiate CPC generation in adult extracardiac fibroblasts using a CRISPR activation system. The induced fibroblasts (>80%) showed phenotypic changes as indicated by an Nkx2.5 cardiac enhancer reporter. The progenitor characteristics were confirmed by colony formation and expression of cardiovascular genes. Cardiac sphere induction segregated the early and late reprogrammed cells that can generate functional cardiomyocytes and vascular cells invitro. Therefore, they were termed CRISPR-induced CPCs (ciCPCs). Transcriptomic analysis showed that cell cycle and heart development pathways were important to accelerate CPC formation during the early reprogramming stage. The CRISPR system opened the silenced chromatin locus, thereby allowing transcriptional factors to access their own promoters and eventually forming a positive feedback loop. The regenerative potential of ciCPCs was assessed after implantation in mouse myocardial infarction models. The engrafted ciCPCs differentiated into cardiovascular cells invivo but also significantly improved contractile function and scar formation. In conclusion, multiplex gene activation was sufficient to drive CPC reprogramming, providing a new cell source for regenerative therapeutics.

Augmenting and directing long-range CRISPR-mediated activation in human cells.

Epigenetic editing is an emerging technology that uses artificial transcription factors (aTFs) to regulate expression of a target gene. Although human genes can be robustly upregulated by targeting aTFs to promoters, the activation induced by directing aTFs to distal transcriptional enhancers is substantially less robust and consistent. Here we show that long-range activation using CRISPR-based aTFs in human cells can be made more efficient and reliable by concurrently targeting an aTF to the target gene promoter. We used this strategy to direct target gene choice for enhancers capable of regulating more than one promoter and to achieve allele-selective activation of human genes by targeting aTFs to SNPs embedded in distally located sequences. Our results broaden the potential applications of the epigenetic editing toolbox for research and therapeutics.

Advances in Enhanced Menaquinone-7 Production From Bacillus subtilis.

The production of nutraceutical compounds through biosynthetic approaches has received considerable attention in recent years. For example, Menaquinone-7 (MK-7), a sub-type of Vitamin K2, biosynthesized from Bacillus subtilis (B. subtilis), proved to be more efficiently produced than the conventional chemical synthesis techniques. This is possible due to the development of B. subtilis as a chassis cell during the biosynthesis stages. Hence, it is imperative to provide insights on the B. subtilis membrane permeability modifications, biofilm reactors, and fermentation optimization as advanced techniques relevant to MK-7 production. Although the traditional gene-editing method of homologous recombination improves the biosynthetic pathway, CRISPR-Cas9 could potentially resolve the drawbacks of traditional genome editing techniques. For these reasons, future studies should explore the applications of CRISPRi (CRISPR interference) and CRISPRa (CRISPR activation) system gene-editing tools in the MK-7 anabolism pathway.

Inducible CRISPRa screen identifies putative enhancers

Enhancers are critical cis-regulatory elements that regulate spatiotemporal gene expression and control cell fates. However, the identification of enhancers in native cellular contexts still remains a challenge. Here, we develop an inducible CRISPR activation (CRISPRa) system by transgenic expression of doxycycline (Dox)-inducible dCas9-VPR in mouse embryonic stem cells (iVPR ESC). With this line, a simple introduction of specific guide RNAs targeting promoters or enhancers allows us to realize the effect of CRISPRa in an inducible, reversible, and Dox concentration-dependent manner. Taking advantage of this system, we induce tiled CRISPRa across genomic regions (105 kilobases) surrounding T (Brachyury), one of the key mesodermal development regulator genes. Moreover, we identify several CRISPRa-responsive elements with chromatin features of putative enhancers, including a region the homologous sequence in which humans harbors a body height risk variant. Genetic deletion of this region in ESC does affect subsequent T gene activation and osteogenic differentiation. Therefore, our inducible CRISPRa ESC line provides a convenient platform for high-throughput screens of putative enhancers.

Deletion of lncRNA XACT does not change expression dosage of X-linked genes, but affects differentiation potential in hPSCs.

Female human pluripotent stem cells (hPSCs) regularly show erosion of X chromosome inactivation featured by the loss of the long non-coding (lnc) RNA XIST and the accumulation of lncXACT. Here, we report that a common mechanism for the initiation of erosion depends on XIST loss but not XACT accumulation on inactive X chromosomes. We further demonstrate that XACT deletion does not affect X-linked gene dosage in eroded hPSCs and that aberrant XIST RNA diffusion induced by the CRISPR activation system is independent of the presence of XACT RNA. In contrast, the deletion of XACT results in the upregulation of neuron-related genes, facilitating neural differentiation in both male and eroded female hPSCs. XACT RNA repression by CRIPSR inhibition results in the same phenotype. Our study finds that XACT is dispensable for maintaining the erosion of X-lined gene repression on inactive X chromosomes but affects neural differentiation in hPSCs.

CRISPR Interference and Activation to Modulate Transcription in Yarrowia lipolytica.

Recent developments in RNA-guided nuclease technologies have advanced the engineering of a wide range of organisms, including the nonconventional yeast Yarrowia lipolytica. Y. lipolytica has been the focus of a range of synthetic biology and metabolic engineering studies due to its high capacity to synthesize and accumulate intracellular lipids. The CRISPR-Cas9 system from Streptococcus pyogenes has been successfully adapted and used for genome editing in Y. lipolytica. However, as engineered strains are moved closer to industrialization, the need for finer control of transcription is still present. To overcome this challenge, we have developed CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems to allow modulating the transcription of endogenous genes. We begin this protocol chapter by describing how to use the CRISPRi system to repress expression of any gene in Y. lipolytica. A second method describes how to use the CRISPRa system to increase expression of native Y. lipolytica genes. Finally, we describe how CRISPRi or CRISPRa vectors can be combined to enable multiplexed activation or repression of more than one gene. The implementation of CRISPRi and CRISPRa systems improves our ability to control gene expression in Y. lipolytica and promises to enable more advanced synthetic biology and metabolic engineering studies in this host.

Kaposi's Sarcoma-Associated Herpesvirus Reactivation by Targeting of a dCas9-Based Transcription Activator to the ORF50 Promoter.

CRISPR activation (CRISPRa) has revealed great potential as a tool to modulate the expression of targeted cellular genes. Here, we successfully applied the CRISPRa system to trigger the Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation in latently infected cells by selectively activating ORF50 gene directly from the virus genome. We found that a nuclease-deficient Cas9 (dCas9) fused to a destabilization domain (DD) and 12 copies of the VP16 activation domain (VP192) triggered a more efficient KSHV lytic cycle and virus production when guided to two different sites on the ORF50 promoter, instead of only a single site. To our surprise, the virus reactivation induced by binding of the stable DD-dCas9-VP192 on the ORF50 promoter was even more efficient than reactivation induced by ectopic expression of ORF50. This suggests that recruitment of additional transcriptional activators to the ORF50 promoter, in addition to ORF50 itself, are needed for the efficient virus production. Further, we show that CRISPRa can be applied to selectively express the early lytic gene, ORF57, without disturbing the viral latency. Therefore, CRISPRa-based systems can be utilized to facilitate virus–host interaction studies by controlling the expression of not only cellular but also of specific KSHV genes.

Abstract 4070: Development of MAEGI as a new class of immune gene therapy for cancer treatment

Abstract Current major types of immunotherapy include checkpoint blockade, adoptive cell transfer, human recombinant cytokines, and cancer vaccines. However, immunotherapy has met challenges in immunologically cold tumors. These challenges urge for new types of immunotherapies that are more potent and potentially less toxic. Recently, we have developed CRISPRa-mediated Multiplexed Activation of Endogenous Genes as an Immunotherapy (MAEGI) (Wang et al. 2019 Nature Immunology, in press). The CRISPR activation (CRISPRa) system uses a catalytically inactive Cas9 (dCas9), enabling simple and flexible gene expression regulation through dCas9-transcriptional activators paired with single guide RNAs (sgRNAs). This enables precise targeting of large gene pools of endogenous genes in a flexible manner. We demonstrate that MAEGI has therapeutic efficacy across three tumor types. Mechanistically, our preliminary work showed that MAEGI treatment elicits anti-tumor immune responses by recruiting effector T cells and remodeling the tumor microenvironment. We will perform advanced development, characterization and optimization of MAEGI, as a novel immune-gene therapy approach to elicit a potent and specific immune response to tumors based on their unique genetic composition. Citation Format: Sidi Chen. Development of MAEGI as a new class of immune gene therapy for cancer treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4070.

Abstract 210: Reprogramming Mouse Fibroblasts Into Cardiac Progenitor Cells Using Crispr Technique Targeting Endogenic Genes

Background and Objective: CRISPR tools that allow for precise manipulation of individual loci have not been used in generation of i nduced c ardiac p rogenitor c ells ( iCPC s). This study was designed to determine the feasibility and effectiveness of reprogramming fibroblasts into iCPC using CRISPR activation (CRISPRa) system. Methods: Tail-tip fibroblasts (TTFs) were isolated from Nkx2-5 cardiac enhancer GFP reporter mice. A gRNA pool targeting 17 progenitor genes was synthesized and transduced with dCas9-VP64 lentivirus into TTFs ( Fig.1A ). The phenotype of iCPCs was then characterized by immunostaining and FACS of progenitor markers. Finally, the cardiac-lineage differentiation potential of iCPCs was determined by immunostaining and electrophysiological assay under defined induction mediums. Results: iCPCs with GFP expression were formed in TTFs after transduction of CRISPRa targeting Isl1, Gata4, Baf60c, Tbx5 and Nkx2-5 (Fig.1B), while GFP was not activated by control virus. Cardiac progenitor markers were activated in iCPCs as shown by immunostaining (Fig.1C). The generation efficiency of Flk1-postive iCPCs induced by CRISPRa was ~60% as showed by FACS. iCPCs can be differentiated into cardiomyocytes as identified by immunostaining of cardiac-specific markers (Fig.1D). The iCPC-derived cardiomyocytes displayed spontaneous beating and showed cardiac action potentials (Fig.1E). Conclusion: The CRISPRa system is an efficient and specific way to generate iCPCs, which could provide a novel source of cells for cardiac regenerative medicine. Conversion of fibroblasts into cardiac progenitors poses a novel therapeutic option for repair of myocardial infarction.