A CRISPR/dCas9 toolkit for functional analysis of maize genes
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ABSTRACT:
SUBMITTER: Gentzel I
PROVIDER: S-EPMC7532566 | biostudies-literature | 2020 Jan
REPOSITORIES: biostudies-literature
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Project description:Reversal in the transcriptional status of desired genes has been exploited for multiple research, therapeutic, and biotechnological purposes. CRISPR/dCas9-based activators can activate transcriptionally silenced genes after being guided by gene-specific gRNA(s). Here, we performed a functional comparison between two such activators, VP64-dCas9-VP64 and dCas9-VP192, in human embryonic kidney cells by the concomitant targeting of POU5F1 and SOX2. We found 22- and 6-fold upregulations in the mRNA level of POU5F1 by dCas9-VP192 and VP64-dCas9-VP64, respectively. Likewise, SOX2 was up-regulated 4- and 2-fold using dCas9-VP192 and VP64dCas9VP64, respectively. For the POU5F1 protein level, we observed 3.7- and 2.2-fold increases with dCas9-VP192 and VP64-dCas9-VP64, respectively. Similarly, the SOX2 expression was 2.4- and 2-fold higher with dCas9-VP192 and VP64-dCas9-VP64, respectively. We also confirmed that activation only happened upon co-transfecting an activator plasmid with multiplex gRNA plasmid with a high specificity to the reference genes. Our data revealed that dCas9-VP192 is more efficient than VP64-dCas9-VP64 for activating reference genes.
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Project description:BackgroundRice (Oryza sativa) is the main food for half of the world's population, and is considered the model for molecular biology studies of monocotyledon species. Although the rice genome was completely sequenced about 15 years ago, the function of most rice genes is still unknown.ResultsIn this study, we developed a vector toolkit that contains 42 vectors for transient expression studies in rice protoplasts and stable expression analysis in transgenic rice. These vectors have been successfully used to study protein subcellular localization, protein-protein interaction, gene overexpression, and the CRISPR/Cas9-mediated gene editing. A novel feature of these vectors is that they contain a universal multiple cloning site, which enables more than 99% of the rice coding sequences to be conveniently transferred between vectors.ConclusionsThe versatile vectors represent a highly efficient and high-throughput toolkit for functional analysis of rice genes.
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Project description:The ability to control the activity of CRISPR-dCas9 with precise spatiotemporal resolution will enable tight genome regulation of user-defined endogenous genes for studying the dynamics of transcriptional regulation. Optogenetic devices with minimal phototoxicity and the capacity for deep tissue penetration are extremely useful for precise spatiotemporal control of cellular behavior and for future clinic translational research. Therefore, capitalizing on synthetic biology and optogenetic design principles, we engineered a far-red light (FRL)-activated CRISPR-dCas9 effector (FACE) device that induces transcription of exogenous or endogenous genes in the presence of FRL stimulation. This versatile system provides a robust and convenient method for precise spatiotemporal control of endogenous gene expression and also has been demonstrated to mediate targeted epigenetic modulation, which can be utilized to efficiently promote differentiation of induced pluripotent stem cells into functional neurons by up-regulating a single neural transcription factor, NEUROG2 This FACE system might facilitate genetic/epigenetic reprogramming in basic biological research and regenerative medicine for future biomedical applications.
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Project description:BackgroundPromoter hypermethylation of tumour suppressor genes is frequently observed during the malignant transformation of colorectal cancer (CRC). However, whether this epigenetic mechanism is functional in cancer or is a mere consequence of the carcinogenic process remains to be elucidated.ResultsIn this work, we performed an integrative multi-omic approach to identify gene candidates with strong correlations between DNA methylation and gene expression in human CRC samples and a set of 8 colon cancer cell lines. As a proof of concept, we combined recent CRISPR-Cas9 epigenome editing tools (dCas9-TET1, dCas9-TET-IM) with a customized arrayed gRNA library to modulate the DNA methylation status of 56 promoters previously linked with strong epigenetic repression in CRC, and we monitored the potential functional consequences of this DNA methylation loss by means of a high-content cell proliferation screen. Overall, the epigenetic modulation of most of these DNA methylated regions had a mild impact on the reactivation of gene expression and on the viability of cancer cells. Interestingly, we found that epigenetic reactivation of RSPO2 in the tumour context was associated with a significant impairment in cell proliferation in p53-/- cancer cell lines, and further validation with human samples demonstrated that the epigenetic silencing of RSPO2 is a mid-late event in the adenoma to carcinoma sequence.ConclusionsThese results highlight the potential role of DNA methylation as a driver mechanism of CRC and paves the way for the identification of novel therapeutic windows based on the epigenetic reactivation of certain tumour suppressor genes.
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Project description:The caption of Fig. 5 and the figure contained an error [1]. The updated figure and caption are published in this correction article.
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Project description:Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems can be engineered as programmable transcription factors to either activate (CRISPRa) or inhibit transcription. Apomixis is extremely valuable for the seed industry in breeding clonal seeds with pure genetic backgrounds. We report here a CRISPR/dCas9-based toolkit equipped with dCas9-VP64 and MS2-p65-HSF1 effectors that may specifically target genes with high activation capability. We explored the application of in vivo CRISPRa targeting of maize BABY BOOM2 (ZmBBM2), acting as a fertilization checkpoint, as a means to engineer parthenogenesis. We detected ZmBBM2 transcripts only in egg cells but not in other maternal gametic cells. Activation of ZmBBM2 in egg cells in vivo caused maternal cell-autonomous parthenogenesis to produce haploid seeds. Our work provides a highly specific gene-activation CRISPRa technology for target cells and verifies its application for parthenogenesis induction in maize.
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Project description:CRISPR-Cas systems provide immunity against mobile genetic elements (MGEs) through sequence-specific targeting by spacer sequences encoded in CRISPR arrays. Spacers are highly variable between microbial strains and can be acquired rapidly, making them well suited for use in strain typing of closely related organisms. However, no tools are currently available to automate the process of reconstructing strain histories using CRISPR spacers. We therefore developed the CRISPR Comparison Toolkit (CCTK) to enable analyses of array relationships. The CCTK includes tools to identify arrays, analyze relationships between arrays using CRISPRdiff and CRISPRtree, and predict targets of spacers. CRISPRdiff visualizes arrays and highlights the similarities between them. CRISPRtree infers a phylogenetic tree from array relationships and presents a hypothesis of the evolutionary history of the arrays. The CCTK unifies several CRISPR analysis tools into a single command line application, including the first tool to infer phylogenies from array relationships.
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Project description:The genetic tractability of the yeast Saccharomyces cerevisiae has made it a key model organism for basic research and a target for metabolic engineering. To streamline the introduction of tagged genes and compartmental markers with powerful Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - CRISPR-associated protein 9 (Cas9)-based genome editing tools, we constructed a Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 toolkit with 3 components: (1) a set of optimized Streptococcus pyogenes Cas9-guide RNA expression vectors with 5 selectable markers and the option to either preclone or cotransform the gRNAs; (2) vectors for the one-step construction of integration cassettes expressing an untagged or green fluorescent protein/red fluorescent protein/hemagglutinin-tagged gene of interest at one of 3 levels, supporting localization and overexpression studies; and (3) integration cassettes containing moderately expressed green fluorescent protein- or red fluorescent protein-tagged compartmental markers for colocalization experiments. These components allow rapid, high-efficiency genomic integrations and modifications with only transient selection for the Cas9 vector, resulting in markerless transformations. To demonstrate the ease of use, we applied our complete set of compartmental markers to colabel all target subcellular compartments with green fluorescent protein and red fluorescent protein. Thus, the MyLO toolkit packages CRISPR-Cas9 technology into a flexible, optimized bundle that allows the stable genomic integration of DNA with the ease of use approaching that of transforming plasmids.
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Project description:High-throughput genetic screens are powerful methods to identify genes linked to a given phenotype. The catalytic null mutant of the Cas9 RNA-guided nuclease (dCas9) can be conveniently used to silence genes of interest in a method also known as CRISPRi. Here, we report a genome-wide CRISPR-dCas9 screen using a starting pool of ~ 92,000 sgRNAs which target random positions in the chromosome of E. coli. To benchmark our method, we first investigate its utility to predict gene essentiality in the genome of E. coli during growth in rich medium. We could identify 79% of the genes previously reported as essential and demonstrate the non-essentiality of some genes annotated as essential. In addition, we took advantage of the intermediate repression levels obtained when targeting the template strand of genes to show that cells are very sensitive to the expression level of a limited set of essential genes. Our data can be visualized on CRISPRbrowser, a custom web interface available at crispr.pasteur.fr. We then apply the screen to discover E. coli genes required by phages ?, T4 and 186 to kill their host, highlighting the involvement of diverse host pathways in the infection process of the three tested phages. We also identify colanic acid capsule synthesis as a shared resistance mechanism to all three phages. Finally, using a plasmid packaging system and a transduction assay, we identify genes required for the formation of functional ? capsids, thus covering the entire phage cycle. This study demonstrates the usefulness and convenience of pooled genome-wide CRISPR-dCas9 screens in bacteria and paves the way for their broader use as a powerful tool in bacterial genomics.
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Project description:The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO, in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases.
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