CRISPR-based Transcriptional Activation Research Articles

Cytotoxicity of Activator Expression in CRISPR-based Transcriptional Activation Systems.

CRISPR-based transcriptional activation (CRISPRa) has extensive research and clinical potential. Here, we show that commonly used CRISPRa systems can exhibit pronounced cytotoxicity. We demonstrate the toxicity of published and new CRISPRa vectors expressing the activation domains (ADs) of the transcription factors p65 and HSF1, components of the synergistic activation mediator (SAM) CRISPRa system. Based on our findings for the SAM system, we extended our studies to additional ADs and the p300 acetyltransferase core domain. We show that the expression of potent transcriptional activators in lentiviral producer cells leads to low lentiviral titers, while their expression in the transduced target cells leads to cell death. Using inducible lentiviral vectors, we could not identify an activator expression window for effective SAM-based CRISPRa without measurable toxicity. The toxicity of current SAM-based CRISPRa systems hinders their wide adoption in biomedical research and introduces selection bottlenecks that may confound genetic screens. Our results suggest that the further development of CRISPRa technology should consider both the efficiency of gene activation and activator toxicity.

Gene-activating nanomedicine for the tumor-oriented infiltration of T cells to enhance immunotherapy against solid tumors

The insufficient infiltration of T cells in solid tumors hinders the therapeutic efficacy of immune checkpoint blockade (ICB) antibodies and chimeric antigen receptor (CAR)-T cells. Promoting T or CAR-T cells to efficiently and specifically migrate into solid tumors is critical for improving current immunotherapies, which remains a challenge. Herein, we proposed a CRISPR-based transcriptional activation (CRISPRa) nanomedicine for mobilizing tumor cells to recruit T cells by activating the expression of T cell chemokines CXC-chemokine ligand (CXCL) 9, CXCL10, and CXCL11. We demonstrated that the CRISPRa nanomedicine could dramatically upregulate the expression of these T cell chemokines in tumor cells, thereby facilitating T cell migration directionally into solid tumors. Intratumoral injection of the CRISPRa nanomedicine inhibited the growth of different solid tumors including melanoma, pancreatic, colonic, and breast cancers. Moreover, tumor-specific versions of the CRISPRa nanomedicine possessing the ability to upregulate the T cell chemokine expression only in tumors rather than normal tissues or cells were constructed. Systemic injection of the tumor-specific CRISPRa nanomedicine specifically recruited T or CAR-T cells into solid tumors and improved the therapeutic effects of ICB antibodies (anti-PD-L1 or anti-CTLA4) and CAR-T cells. Thus, the gene-activating nanomedicine provides a promising strategy for enhancing T cell infiltration to improve current immunotherapies against solid tumors.

Target Gene Identification and sgRNA Design for Waterlogging Tolerance in Foxtail Millet via CRISPR-Based Transcriptional Activation

Background: CRISPR activation (CRISPRa) uses non-functional Cas9 endonuclease (dCas9) but retains the genome targeting ability through its single guide RNAs (sgRNAs). CRISPRa is widely used as a gene activation system exploiting its ability in recruiting various transcriptional activation domains (TADs) for enhancing expression of the target gene(s). Drought tolerant and resource-efficient crops like millets have the potential for mitigating effects of climate change and for enhancing food security. Objective: This study aimed to use the Setaria italica (foxtail millet) genome sequence in the identification of a target gene and the subsequent generation of sgRNAs for use in CRISPRa for conferring water logging tolerance that will benefit the future expansion of its cultivation area. Methods and Results: Leveraging existing RNA-seq data and information on functional studies in model plants and from other cereal species, maize and barley, have enabled the identification of candidate ERFVII from the foxtail millet genome sequence in the attempt to engineer water logging tolerance. The study provides a step-by-step example for using publicly accessible databases and bioinformatics tools from NCBI and Phytozome for the identification and characterization of the ortholog from Setaria italica. Soft berry was used for promoter annotation to obtain the transcription start site (TSS). Subsequently, CRISP-P 2.0 design tools were employed to generate and select a few efficient sgRNAs for CRISPRa that minimize potentially deleterious off-target binding. Conclusion: The study is a useful example of how to advance in genomics research including the revolutionizing CRISPR technology in Setaria italica, which can be adopted in other plant species, through the utilization of the available genome sequence.

Tissue-specific activation of gene expression by the Synergistic Activation Mediator (SAM) CRISPRa system in mice

CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.

A viral guide RNA delivery system for CRISPR-based transcriptional activation and heritable targeted DNA demethylation in Arabidopsis thaliana.

Plant RNA viruses are used as delivery vectors for their high level of accumulation and efficient spread during virus multiplication and movement. Utilizing this concept, several viral-based guide RNA delivery platforms for CRISPR-Cas9 genome editing have been developed. The CRISPR-Cas9 system has also been adapted for epigenome editing. While systems have been developed for CRISPR-Cas9 based gene activation or site-specific DNA demethylation, viral delivery of guide RNAs remains to be developed for these purposes. To address this gap we have developed a tobacco rattle virus (TRV)-based single guide RNA delivery system for epigenome editing in Arabidopsis thaliana. Because tRNA-like sequences have been shown to facilitate the cell-to-cell movement of RNAs in plants, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome to promote heritable epigenome editing. We demonstrate that the tRNA-gRNA system with TRV can be used for both transcriptional activation and targeted DNA demethylation of the FLOWERING WAGENINGEN gene in Arabidopsis. We achieved up to ~8% heritability of the induced demethylation phenotype in the progeny of virus inoculated plants. We did not detect the virus in the next generation, indicating effective clearance of the virus from plant tissues. Thus, TRV delivery, combined with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing.

Gene Regulatory Network Analysis and Engineering Directs Development and Vascularization of Multilineage Human Liver Organoids.

SUMMARYPluripotent stem cell (PSC)-derived organoids have emerged as novel multicellular models of human tissue development but display immature phenotypes, aberrant tissue fates, and a limited subset of cells. Here, we demonstrate that integrated analysis and engineering of gene regulatory networks (GRNs) in PSC-derived multilineage human liver organoids direct maturation and vascular morphogenesis in vitro. Overexpression of PROX1 and ATF5, combined with targeted CRISPR-based transcriptional activation of endogenous CYP3A4, reprograms tissue GRNs and improves native liver functions, such as FXR signaling, CYP3A4 enzymatic activity, and stromal cell reactivity. The engineered tissues possess superior liver identity when compared with other PSC-derived liver organoids and show the presence of hepatocyte, biliary, endothelial, and stellate-like cell populations in single-cell RNA-seq analysis. Finally, they show hepatic functions when studied in vivo. Collectively, our approach provides an experimental framework to direct organogenesis in vitro by systematically probing molecular pathways and transcriptional networks that promote tissue development.

Gain-of-function studies with CRISPR-based transcriptional activation at endogenous genomic loci reveals genes with critical roles for myeloma cells

Gain-of-function studies with CRISPR-based transcriptional activation at endogenous genomic loci reveals genes with critical roles for myeloma cells

A Neuron-Optimized CRISPR/dCas9 Activation System for Robust and Specific Gene Regulation.

CRISPR-based technology has provided new avenues to interrogate gene function, but difficulties in transgene expression in post-mitotic neurons has delayed incorporation of these tools in the central nervous system (CNS). Here, we demonstrate a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation (CRISPRa) system capable of robust, modular, and tunable gene induction and multiplexed gene regulation across several primary rodent neuron culture systems. CRISPRa targeting unique promoters in the complex multi-transcript gene brain-derived neurotrophic factor (Bdnf) revealed both transcript- and genome-level selectivity of this approach, in addition to highlighting downstream transcriptional and physiological consequences of Bdnf regulation. Finally, we illustrate that CRISPRa is highly efficient in vivo, resulting in increased protein levels of a target gene in diverse brain structures. Taken together, these results demonstrate that CRISPRa is an efficient and selective method to study gene expression programs in brain health and disease.

CRISPR Activation Screen for Drivers of MM Cell Proliferation

CRISPR/Cas9-based gene editing has become a powerful tool for loss-of-function (LOF) studies and has allowed us to systematically interrogate the function of genes regulating the survival and proliferation of multiple myeloma (MM) cells in vitro, in vivo and in the context of treatment resistance (e.g. De Matos Simoes et al., Shirasaki et al., and Gandolfi et al. ASH 2017). We reasoned, however, that important additional information can be obtained from CRISPR-based gain-of-function (GOF) approaches which can achieve transcriptional activation at endogenous genomic loci. We thus performed genome-scale CRISPR activation studies using the dCas9-P65-HSF transcriptional activation system (in which a Cas9 variant lacking nuclease activity [dCas9] confers P65-HSF-mediated activation of genes recognized by sgRNAs against their promoter regions). Specifically, MM.1S cells were transduced with the dCas9-P65-HSF system and pooled lentiviral particles of the Calabrese CRISPR activation sgRNA library, consisting of 2 pooled sgRNA sub-libraries (total of ~110,000 sgRNAs targeting ~18000 genes, at initial coverage of 800 cells/sgRNA). Cells were cultured for 12 weeks and harvested at baseline and various intervals, e.g. 4 and 12 weeks of culture. Next generation sequencing of genomic DNA quantified the abundance of sgRNAs in the tumor cell population and genes were ranked (with rank aggregation algorithms) in terms of their sgRNA enrichment or depletion. These analyses allowed us to observe a series of genes with statistically significant sgRNA enrichment and known or presumed roles in MM biology, including key MM transcription factors such as IRF4, the thalidomide derivative targets IKZF3 and IKZF1, and the co-factor POU2AF1; known oncogenes, e.g. KRAS and MYC; NF-kappaB pathway members, e.g. RELA; and signal transduction regulators, e.g. IGF1R and its downstream effectors IRS1 and AKT2. These results are consistent with the major depletion of sgRNAs for these genes in loss-of-function (LOF) CRISPR knockout studies. However, several other genes with significant sgRNA enrichment in CRISPR activation studies did not exhibit major sgRNA depletion in CRISPR knockout studies, including the B/plasma cell transcription factor POU2F2 (Oct2), for which high protein expression correlates with reduced survival in MM (Toman I. et al 2011); the transcription factor PAX2, the TRAF interacting protein TIFA, or the Toll-like receptor TLR4. Interestingly, significant depletion of sgRNAs was observed for several genes with known or proposed tumor suppressive properties including YAP1 (an oncogene for solid tumors, but reported as tumor suppressor for MM and other blood cancers); the pro-death TNFRSF10A (TRAIL receptor DR4), TP73, CDKN1A, the negative regulator of c-Myc MXI1, or the pro-apoptotic Bcl2 family member BAK1. Depletion or enrichment of sgRNAs for most of the aforementioned genes was detectable by 4 weeks of culture, while more pronounced changes and detection of additional statistically-significant hits was observed in later time-points. For genes with significant sgRNA enrichment in our CRISPR activation study, we examined a series of molecular alterations, including transcript overexpression in MM cell lines or patient-derived samples vs. normal plasma cells, or relapsed/refractory MM vs. earlier disease MM stages; mutational status; correlation of transcript levels with clinical outcome in MM; and extent of open chromatin (based on H3K27Ac chromatin marks) within or proximal to each gene in MM cell lines. Some “hits” from our screen exhibited at least one of these molecular alterations, but most genes harbored no such alterations or their magnitude or frequency ranked outside the top 50-100 genes. These results suggest that CRISPR activation studies can identify important putative regulators of MM biology, which may not be readily detectable based on known annotations of the MM cell genome, transcriptome, or epigenome. Genome-scale CRISPR-based transcriptional activation are an important gain-of-function system to uncover genes which induce vs. suppress tumor cell survival and proliferation, and provide information orthogonal to those yielded by other CRISPR-based approaches that involve loss-of-function interventions. Our use of CRISPR activation allowed us to both validate previously known genes and identify promising new candidate regulators of MM cell biology. DisclosuresMitsiades:EMD Serono: Research Funding; Abbvie: Research Funding; Takeda: Other: employment of a relative; TEVA: Research Funding; Janssen/ Johnson & Johnson: Research Funding.