Ontology highlight
ABSTRACT:
PROVIDER: PRJNA977471 | ENA |
REPOSITORIES: ENA
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Project description:Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technologies have evolved rapidly over the past decade with the continuous discovery of new Cas systems. In particular, RNA-targeting CRISPR-Cas13 proteins are promising single-effector systems to regulate target mRNAs without altering genomic DNA, yet the current Cas13 systems are restrained by suboptimal efficiencies. Here, we show that U1 promoter-driven CRISPR RNAs (crRNAs) increase the efficiency of various applications, including RNA knockdown and editing, without modifying the Cas13 protein effector. We confirm that U1-driven crRNAs are exported into the cytoplasm, while conventional U6 promoter-driven crRNAs are mostly confined to the nucleus. Furthermore, we reveal that the end positions of crRNAs expressed by the U1 promoter are consistent regardless of guide sequences and lengths. We also demonstrate that U1-driven crRNAs, but not U6-driven crRNAs, can efficiently repress the translation of target genes in combination with catalytically inactive Cas13 proteins. Finally, we show that U1-driven crRNAs can counteract the inhibitory effect of miRNAs. Our simple and effective engineering enables unprecedented cytosolic RNA-targeting applications.
2024-09-15 | GSE275245 | GEO
Project description:The CCR4-NOT complex is a central regulator of gene expression, orchestrating mRNA destabilization and decay through interactions with RNA-binding proteins (RBPs) and the miRNA-induced silencing complex (miRISC). However, identifying which RBP and miRNA target sites actively recruit this complex to engender mRNA turnover remains challenging, due in part to the multiple modes by which the CCR4-NOT can be recruited to targets. Here, to address this, we developed TRACER (Targeted RNA Association of CCR4-NOT and Element Recovery), a crosslinking approach that enables high-throughput mapping of cis-acting RNA elements that recruit the CCR4-NOT complex to target RNAs. TRACER analysis of a human epithelial cell line uncovers thousands of CCR4-NOT target elements, including many of which map to known and/or predicted RBP binding motifs, as well as miRNA target sites. Importantly, we show that CCR4-NOT recruitment elements identified by TRACER play important roles in promoting mRNA decay. Taken together, our approach provides the first high-resolution transcriptome-wide map of CCR4-NOT targeting elements in human cells that engender mRNA decay.
2026-04-13 | GSE302649 | GEO
Project description:CRISPR-Cas13 systems have been adapted as versatile toolkits for RNA-related applications. Here we systematically evaluate the performance of several prominent Cas13 family effectors (Cas13a, Cas13b and Cas13d) under lentiviral vectors and reveal surprisingly differential defects and characteristics of these systems. Using RNA immunoprecipitation sequencing, transcriptome profiling, biochemistry analysis and high-throughput CRISPR-Cas13 screening approaches, we determine that each Cas13 system has its intrinsic RNA targets in mammalian cells. Viral process-related host genes can be targeted by Cas13 and affect the production of fertile lentiviral particles, thereby restricting the utility of lentiviral Cas13 systems. Multiple RNase activities of Cas13 are involved in endogenous RNA targeting. Unlike target-induced collateral effect, intrinsic RNA targeting can be specific, target-independent and dynamically tuned by varied states of Cas13 nucleases. Our work not only provides guidance to appropriately utilize lentiviral Cas13 systems, but also raises cautions about intrinsic RNA targeting during Cas13-based basic and therapeutic applications.
2023-10-09 | GSE202898 | GEO
Project description:CRISPR-Cas13 systems have been adapted as versatile toolkits for RNA-related applications. Here we systematically evaluate the performance of several prominent Cas13 family effectors (Cas13a, Cas13b and Cas13d) under lentiviral vectors and reveal surprisingly differential defects and characteristics of these systems. Using RNA immunoprecipitation sequencing, transcriptome profiling, biochemistry analysis and high-throughput CRISPR-Cas13 screening approaches, we determine that each Cas13 system has its intrinsic RNA targets in mammalian cells. Viral process-related host genes can be targeted by Cas13 and affect the production of fertile lentiviral particles, thereby restricting the utility of lentiviral Cas13 systems. Multiple RNase activities of Cas13 are involved in endogenous RNA targeting. Unlike target-induced collateral effect, intrinsic RNA targeting can be specific, target-independent and dynamically tuned by varied states of Cas13 nucleases. Our work not only provides guidance to appropriately utilize lentiviral Cas13 systems, but also raises cautions about intrinsic RNA targeting during Cas13-based basic and therapeutic applications.
2023-10-09 | GSE202841 | GEO
Project description:CRISPR-Cas13 systems have been adapted as versatile toolkits for RNA-related applications. Here we systematically evaluate the performance of several prominent Cas13 family effectors (Cas13a, Cas13b and Cas13d) under lentiviral vectors and reveal surprisingly differential defects and characteristics of these systems. Using RNA immunoprecipitation sequencing, transcriptome profiling, biochemistry analysis and high-throughput CRISPR-Cas13 screening approaches, we determine that each Cas13 system has its intrinsic RNA targets in mammalian cells. Viral process-related host genes can be targeted by Cas13 and affect the production of fertile lentiviral particles, thereby restricting the utility of lentiviral Cas13 systems. Multiple RNase activities of Cas13 are involved in endogenous RNA targeting. Unlike target-induced collateral effect, intrinsic RNA targeting can be specific, target-independent and dynamically tuned by varied states of Cas13 nucleases. Our work not only provides guidance to appropriately utilize lentiviral Cas13 systems, but also raises cautions about intrinsic RNA targeting during Cas13-based basic and therapeutic applications.
2023-10-09 | GSE202838 | GEO
Project description:APEX2 fused to RNA binding proteins MS2 or an engineered Cas13
2020-12-11 | MSV000086581 | MassIVE
Project description:The CCR4-NOT complex is a central regulator of gene expression, orchestrating mRNA destabilization and decay through interactions with RNA-binding proteins (RBPs) and the miRNA-induced silencing complex (miRISC). However, identifying which RBP and miRNA target sites actively recruit this complex to engender mRNA turnover remains challenging, due in part to the multiple modes by which the CCR4-NOT can be recruited to targets. Here, to address this, we developed TRACER (Targeted RNA Association of CCR4-NOT and Element Recovery), a crosslinking approach that enables high-throughput mapping of cis-acting RNA elements that recruit the CCR4-NOT complex to target RNAs. TRACER analysis of a human epithelial cell line uncovers thousands of CCR4-NOT target elements, including many of which map to known and/or predicted RBP binding motifs, as well as miRNA target sites. Importantly, we show that CCR4-NOT recruitment elements identified by TRACER play important roles in promoting mRNA decay. Taken together, our approach provides the first high-resolution transcriptome-wide map of CCR4-NOT targeting elements in human cells that engender mRNA decay.
2026-04-13 | GSE302648 | GEO
Project description:RNA-targeting CRISPR-Cas13 enzymes are robust RNA knockdown tools with both on-target and collateral cleavage activities. However, to this date, the in vivo RNA cleavage mechanisms remain poorly understood. Here, we combine in vitro and in vivo methods to elucidate the exact cleavage sites of Cas13. We reveal that some subtypes of Cas13, including Cas13b and Cas13bt, cleave the target RNA in predominant positions, and rational engineering of Cas13 further improves the precision. Building on this fact, we developed RNA segment editing (RSE), a targeted RNA cleavage and repair method, to restore dysfunctional RNA in cells. We anticipate that RSE will enable precision RNA engineering for therapeutics and basic research.
2026-03-02 | GSE313509 | GEO