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Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): synthetic construct Homo sapiens

PROVIDER: GSE202899 | GEO | 2023/10/09

REPOSITORIES: GEO

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Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems [Screen]

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

Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems [RNA-seq]

2023-10-09 | GSE202838 | GEO

Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems [RIP-seq]

2023-10-09 | GSE202898 | GEO

RNA targeting with CRISPR-Cas13.

Project description:RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference can efficiently knockdown RNAs, but it is prone to off-target effects, and visualizing RNAs typically relies on the introduction of exogenous tags. Here we demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR-Cas effector Cas13a (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.

| S-EPMC5706658 | biostudies-literature

A target expression threshold dictates invader defense and autoimmunity by CRISPR-Cas13

Project description:This SuperSeries is composed of the SubSeries listed below.

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CRISPR Display: A modular method for locus-specific targeting of long noncoding RNAs and synthetic RNA devices in vivo

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2014-03-31 | E-GEOD-50025 | biostudies-arrayexpress

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Project description:This SuperSeries is composed of the SubSeries listed below.

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