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Transcriptional response to collateral RNA cleavage by LbuCas13a

ABSTRACT: Transcriptional response to collateral RNA cleavage by LbuCas13a

PROVIDER: PRJNA911286 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR22689964.fastq.gz	Fastqsanger.gz
	SRR22689965.fastq.gz	Fastqsanger.gz
	SRR22689966.fastq.gz	Fastqsanger.gz
	SRR22689967.fastq.gz	Fastqsanger.gz
	SRR22689968.fastq.gz	Fastqsanger.gz

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Transcriptional response to collateral RNA cleavage by LbuCas13a

Project description:LbuCas13a exhibits widespread collateral RNA cleavage in human cells after activation by a specific target RNA. We wanted to explore the cells response to this collateral RNA cleavage. Therefore we performed RNA-seq at 16 hours after RNP transduction. From prior experiments we know that at this timepoint RNA integrity has largely recovered and cells are entering apoptosis.

2023-01-19 | GSE220759 | GEO

Temporal dynamics of collateral RNA cleavage by LbuCas13a in human cells

Project description:We have found that LbuCas13a exhibits collateral RNA cleavage activity in human cells after activation by the target RNA. Here we use total RNA sequencing to explore which RNAs are depleted by the collateral activity of LbuCas13a. ERCC spike-in RNAs were used for normalization between samples. This revealed near global depletion of mRNAs at 50 and 200 minutes after Cas13 protein and guide delivery. Mitochondrial and nuclear transcripts were not affected.

2025-09-24 | GSE308909 | GEO

Temporal dynamics of collateral RNA cleavage by LbuCas13a in human cells

Project description:Temporal dynamics of collateral RNA cleavage by LbuCas13a in human cells

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transcriptome-wide collateral effects of Cas13d variants

Project description:The intrinsic collateral cleavage activity of Cas13d,which refers to the gRNA-independent degradation of bystander RNA, significantly limits its therapeutic potential. To extensively evaluate the collateral effects of hpCas13d genome-wide, we conducted RNA-seq analysis of the transcriptome in HEK293T cells after the transfections with dCas13d, wtCas13d, hpCas13d, and hfCas13d . Compared to dCas13d, wtCas13d with a PPIA gRNA (reported to induce the significant collateral cleavage) resulted in significant downregulations of thousands genes. In sharp contrast, hpCas13d and hfCas13d showed much less off-target genes, respectively. These findings demonstrate that hpCas13d exhibits reduced collateral activity, rendering it suitable for in vivo applications.

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Characterizing the Collateral Activity of CRISPR/Cas13 in Mammalian Cells: Implications for RNA Editing and Therapeutic Applications

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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Project description:Cas13 is a unique family of CRISPR endonucleases exhibiting programmable binding and cleavage of RNAs and is a strong candidate for eukaryotic RNA knockdown in the laboratory and the clinic. However, sequence-specific binding of Cas13 to the target RNA unleashes non-specific bystander RNA cleavage, or collateral activity, which may confound knockdown experiments and raises concerns for therapeutic applications. Although conserved across orthologs and robust in cell-free and bacterial environments, the extent of collateral activity in mammalian cells remains disputed. Here, we investigate Cas13d collateral activity in the context of an RNA-targeting therapy for myotonic dystrophy type 1, a disease caused by a transcribed long CTG repeat expansion. We find that when targeting CUGn RNA in HeLa and other cell lines, Cas13d depletes endogenous and transgenic RNAs, interferes with critical cellular processes, and activates stress response and apoptosis pathways. We also observe collateral effects when targeting other repetitive and unique transgenic sequences, and we provide evidence for collateral activity when targeting highly expressed endogenous transcripts. To minimize collateral activity for repeat-targeting Cas13d therapeutics, we introduce gRNA excision for negative-autoregulatory optimization (GENO), a simple strategy that leverages crRNA processing to control Cas13d expression and is easily integrated into an AAV gene therapy. We argue that thorough assessment of collateral activity is necessary when applying Cas13d in mammalian cells and that implementation of GENO illustrates the advantages of compact and universally robust regulatory systems for Cas-based gene therapies.

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