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

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

PROVIDER: PRJNA837526 | ENA |

REPOSITORIES: ENA

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Dataset's files

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			Action	DRS
	SRR19176131_1.fastq.gz	Fastqsanger.gz
	SRR19176131_2.fastq.gz	Fastqsanger.gz
	SRR19176132_1.fastq.gz	Fastqsanger.gz
	SRR19176132_2.fastq.gz	Fastqsanger.gz
	SRR19176133_1.fastq.gz	Fastqsanger.gz

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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

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

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Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

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

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Project description:We used RNA-seq to detect genome-wide gene expression change when a reporter RNA was targeted by Cas13

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Project description:The CRISPR/Cas13 system has garnered attention as a potential tool for RNA editing. However, the degree of collateral activity among various Cas13 orthologs and their cytotoxic effects in mammalian cells remain contentious, potentially impacting their applications. In this study, we observed differential collateral activities for LwaCas13a and RfxCas13d in 293T and U87 cells by applying both sensitive dual-fluorescence (mRuby/GFP) reporter and quantifiable dual-luciferase (Fluc/Rluc) reporter, with LwaCas13a displaying notable activity contrary to previous reports. However, significant collateral RNA cleavage exerted only a modest impact on cell viability. Furthermore, the collateral activity of LwaCas13a mildly impeded but did not arrest, porcine embryo development. Our findings reveal that distinct collateral RNA cleavage by Cas13 slightly suppresses mammalian cell proliferation and embryo development. This could account for the lack of reported collateral effects in numerous prior studies and offers new insights into the implications of the collateral activity of Cas13 for clinical application.

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