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Reiterative Enrichment and Authentication of CRISPRi Targets (REACT) identifies the proteasome as a key contributor to HIV-1 latency.
The establishment of HIV-1 latency gives rise to persistent chronic infection that requires life-long treatment. To reverse latency for viral eradiation, the HIV-1 Tat protein and its associated ELL2-containing Super Elongation Complexes (ELL2-SECs) are essential to activate HIV-1 transcription. Despite efforts to identify effective latency-reversing agents (LRA), avenues for exposing latent HIV-1 remain inadequate, prompting the need to identify novel LRA targets. Here, by conducting a CRISPR interference-based screen to reiteratively enrich loss-of-function genotypes that increase HIV-1 transcription in latently infected CD4+ T cells, we have discovered a key role of the proteasome in maintaining viral latency. Downregulating or inhibiting the proteasome promotes Tat-transactivation in cell line models. Furthermore, the FDA-approved proteasome inhibitors bortezomib and carfilzomib strongly synergize with existing LRAs to reactivate HIV-1 in CD4+ T cells from antiretroviral therapy-suppressed individuals without inducing cell activation or proliferation. Mechanistically, downregulating/inhibiting the proteasome elevates the levels of ELL2 and ELL2-SECs to enable Tat-transactivation, indicating the proteasome-ELL2 axis as a key regulator of HIV-1 latency and promising target for therapeutic intervention.
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Tumor cell-targeted delivery of CRISPR/Cas9 by aptamer-functionalized lipopolymer for therapeutic genome editing of VEGFA in osteosarcoma.
Osteosarcoma (OS) is a highly aggressive pediatric cancer, characterized by frequent lung metastasis and pathologic bone destruction. Vascular endothelial growth factor A (VEGFA), highly expressed in OS, not only contributes to angiogenesis within the tumor microenvironment via paracrine stimulation of vascular endothelial cells, but also acts as an autocrine survival factor for tumor cell themselves, thus making it a promising therapeutic target for OS. CRISPR/Cas9 is a versatile genome editing technology and holds tremendous promise for cancer treatment. However, a major bottleneck to achieve the therapeutic potential of the CRISPR/Cas9 is the lack of in vivo tumor-targeted delivery systems. Here, we screened an OS cell-specific aptamer (LC09) and developed a LC09-functionalized PEG-PEI-Cholesterol (PPC) lipopolymer encapsulating CRISPR/Cas9 plasmids encoding VEGFA gRNA and Cas9. Our results demonstrated that LC09 facilitated selective distribution of CRISPR/Cas9 in both orthotopic OS and lung metastasis, leading to effective VEGFA genome editing in tumor, decreased VEGFA expression and secretion, inhibited orthotopic OS malignancy and lung metastasis, as well as reduced angiogenesis and bone lesion with no detectable toxicity. The delivery system simultaneously restrained autocrine and paracrine VEGFA signaling in tumor cells and could facilitate translating CRISPR-Cas9 into clinical cancer treatment.
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In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.
In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer (CRC) and metastasis, which rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer-predisposing mutations. Autochthonous tumor formation is induced by CRISPR-Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/Δ;KrasG12D/+;Trp53Δ/Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5+ stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.
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Defining genome-wide CRISPR-Cas genome-editing nuclease activity with GUIDE-seq.
Genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) is a sensitive, unbiased, genome-wide method for defining the activity of genome-editing nucleases in living cells. GUIDE-seq is based on the principle of efficient integration of an end-protected double-stranded oligodeoxynucleotide tag into sites of nuclease-induced DNA double-stranded breaks, followed by amplification of tag-containing genomic DNA molecules and high-throughput sequencing. Here we describe a detailed GUIDE-seq protocol including cell transfection, library preparation, sequencing and bioinformatic analysis. The entire protocol including cell culture can be completed in 9 d. Once tag-integrated genomic DNA is isolated, library preparation, sequencing and analysis can be performed in 3 d. The result is a genome-wide catalog of off-target sites ranked by nuclease activity as measured by GUIDE-seq read counts. GUIDE-seq is one of the most sensitive cell-based methods for defining genome-wide off-target activity and has been broadly adopted for research and therapeutic use.
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Use of the CRISPR-Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes.
The CRISPR-Cas9 system makes it possible to cause double-strand breaks in specific regions, inducing repair. In the presence of a donor construct, repair can involve insertion or 'knock-in' of an exogenous cassette. One common application of knock-in technology is to generate cell lines expressing fluorescently tagged endogenous proteins. The standard approach relies on production of a donor plasmid with ∼500 to 1000 bp of homology on either side of an insertion cassette that contains the fluorescent protein open reading frame (ORF). We present two alternative methods for knock-in of fluorescent protein ORFs into Cas9-expressing Drosophila S2R+ cultured cells, the single-stranded DNA (ssDNA) Drop-In method and the CRISPaint universal donor method. Both methods eliminate the need to clone a large plasmid donor for each target. We discuss the advantages and limitations of the standard, ssDNA Drop-In, and CRISPaint methods for fluorescent protein tagging in Drosophila cultured cells. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Knock-in into Cas9-positive S2R+ cells using the ssDNA Drop-In approach Basic Protocol 2: Knock-in into Cas9-positive S2R+ cells by homology-independent insertion of universal donor plasmids that provide mNeonGreen (CRISPaint method) Support Protocol 1: sgRNA design and cloning Support Protocol 2: ssDNA donor synthesis Support Protocol 3: Transfection using Effectene Support Protocol 4: Electroporation of S2R+-MT::Cas9 Drosophila cells Support Protocol 5: Single-cell isolation of fluorescent cells using FACS.
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CRISPR-Cas9 genome engineering of primary CD4+ T cells for the interrogation of HIV-host factor interactions.
CRISPR-Cas9 gene-editing strategies have revolutionized our ability to engineer the human genome for robust functional interrogation of complex biological processes. We have recently adapted this technology for use in primary human CD4+ T cells to create a high-throughput platform for analyzing the role of host factors in HIV infection and pathogenesis. Briefly, CRISPR-Cas9 ribonucleoproteins (crRNPs) are synthesized in vitro and delivered to activated CD4+ T cells by nucleofection. These cells are then assayed for editing efficiency and expanded for use in downstream cellular, genetic, or protein-based assays. This platform supports the rapid, arrayed generation of multiple gene manipulations and is widely adaptable across culture conditions, infection protocols, and downstream applications. Here, we present detailed protocols for crRNP synthesis, primary T-cell culture, 96-well nucleofection, molecular validation, and HIV infection, and discuss additional considerations for guide and screen design, as well as crRNP multiplexing. Taken together, this procedure allows high-throughput identification and mechanistic interrogation of HIV host factors in primary CD4+ T cells by gene knockout, validation, and HIV spreading infection in as little as 2-3 weeks.
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Suppression of Epstein-Barr virus DNA load in latently infected nasopharyngeal carcinoma cells by CRISPR/Cas9.
Epstein-Barr virus (EBV) infects more than 90% of the world's adult population. Once established, latent infection of nasopharyngeal epithelial cells with EBV is difficult to eradicate and might lead to the development of nasopharyngeal carcinoma (NPC) in a small subset of individuals. In this study we explored the anti-EBV potential of CRISPR/Cas9 targeting of EBV genome in infected NPC cells. We designed gRNAs to target different regions of the EBV genome and transfected them into C666-1 cells. The levels of EBV DNA in transfected cells were decreased by about 50%. The suppressive effect on EBV DNA load lasted for weeks but could not be further enhanced by re-transfection of gRNA. Suppression of EBV by CRISPR/Cas9 did not affect survival of C666-1 cells but sensitized them to chemotherapeutic killing by cisplatin and 5-fluorouracil. Our work provides the proof-of-principle for suppressing EBV DNA load with CRISPR/Cas9 and a potential new strategy to sensitize EBV-infected NPC cells to chemotherapy.
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Selection on the regulation of sympathetic nervous activity in humans and chimpanzees.
Adrenergic α2C receptor (ADRA2C) is an inhibitory modulator of the sympathetic nervous system. Knockout mice for this gene show physiological and behavioural alterations that are associated with the fight-or-flight response. There is evidence of positive selection on the regulation of this gene during chicken domestication. Here, we find that the neuronal expression of ADRA2C is lower in human and chimpanzee than in other primates. On the basis of three-dimensional chromatin structure, we identified a cis-regulatory region whose DNA sequences have been significantly accelerated in human and chimpanzee. Active histone modification marks this region in rhesus macaque but not in human and chimpanzee; instead, repressive marks are enriched in various human brain samples. This region contains two neuron-restrictive silencer factor (NRSF) binding motifs, each of which harbours a polymorphism. Our genotyping and analysis of population genome data indicate that at both polymorphic sites, the derived allele has reached fixation in humans and chimpanzees but not in bonobos, whereas only the ancestral allele is present among macaques. Our CRISPR/Cas9 genome editing and reporter assays show that both derived nucleotides repress ADRA2C, most likely by increasing NRSF binding. In addition, we detected signatures of recent positive selection for lower neuronal ADRA2C expression in humans. Our findings indicate that there has been selective pressure for enhanced sympathetic nervous activity in the evolution of humans and chimpanzees.
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TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus.
Successful RNAi applications depend on strategies allowing robust and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. Here, we propose a novel avenue which is integration of a promoterless shmiRNA, i.e. a shRNA embedded in a micro-RNA (miRNA) scaffold, into an engineered genomic miRNA locus. For proof-of-concept, we used TALE or CRISPR/Cas9 nucleases to site-specifically integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr locus in hepatoma cells, with the aim to obtain cellular clones that are genetically protected against HCV infection. Using reporter assays, Northern blotting and qRT-PCR, we confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality in selected cellular progeny. Moreover, we employed a comprehensive battery of PCR, cDNA/miRNA profiling and whole genome sequencing analyses to validate targeted integration of a single shmiRNA molecule at the expected position, and to rule out deleterious effects on the genomes or transcriptomes of the engineered cells. Importantly, a subgenomic HCV replicon and a full-length reporter virus, but not a Dengue virus control, were significantly impaired in the modified cells. Our original combination of DNA engineering and RNAi expression technologies benefits numerous applications, from miRNA, genome and transgenesis research, to human gene therapy.
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Increased Cytotoxicity of Herpes Simplex Virus Thymidine Kinase Expression in Human Induced Pluripotent Stem Cells.
Human induced pluripotent stem cells (iPSCs) hold enormous promise for regenerative medicine. The major safety concern is the tumorigenicity of transplanted cells derived from iPSCs. A potential solution would be to introduce a suicide gene into iPSCs as a safety switch. The herpes simplex virus type 1 thymidine kinase (HSV-TK) gene, in combination with ganciclovir, is the most widely used enzyme/prodrug suicide system from basic research to clinical applications. In the present study, we attempted to establish human iPSCs that stably expressed HSV-TK with either lentiviral vectors or CRISPR/Cas9-mediated genome editing. However, this task was difficult to achieve, because high-level and/or constitutive expression of HSV-TK resulted in the induction of cell death or silencing of HSV-TK expression. A nucleotide metabolism analysis suggested that excessive accumulation of thymidine triphosphate, caused by HSV-TK expression, resulted in an imbalance in the dNTP pools. This unbalanced state led to DNA synthesis inhibition and cell death in a process similar to a "thymidine block", but more severe. We also demonstrated that the Tet-inducible system was a feasible solution for overcoming the cytotoxicity of HSV-TK expression. Our results provided a warning against using the HSV-TK gene in human iPSCs, particularly in clinical applications.
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