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Temporal dynamics of collateral RNA cleavage by LbuCas13a in human cells

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE308909 | GEO | 2025/09/24

REPOSITORIES: GEO

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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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Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells

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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Substantial collateral effects induced by CasRx in mammalian cells and mice

Project description:CRISPR-Cas13 systems have recently been employed to silence RNAs in yeast, plants, mammalian cell lines and animals, however, collateral effects induced by Cas13 may impede their research and therapeutic applications. Here, we found Cas13a and CasRx-mediated RNA knockdown induced substantial collateral effects on both exogenous and endogenous genes in mammalian cells. Moreover, transgenic mice carrying CasRx, with or without targeting guide RNA, exhibited high lethality. Therefore, our results present limitations of CasRx, requiring a solution to address its fidelity before clinical applications.

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Project description:We found that several variants with 2-4 mutations on Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains showed undiminished on-target activity but markedly reduced collateral effects. Furthermore, transcriptome-wide off-target effects and cell growth arrest induced by wild-type Cas13d were found to be absent for a Cas13d variant. Thus, high-fidelity Cas13 variants with minimal collateral effects are now available for the targeted degradation of RNAs in basic research and therapeutic applications.

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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.

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Transcriptional response to collateral RNA cleavage by LbuCas13a

Project description:Transcriptional response to collateral RNA cleavage by LbuCas13a

| PRJNA911286 | ENA

Proteomic analysis reveals the high specificity and lack of collateral activity of targeted PspCas13b

Project description:To gain a global view on the impact of the collateral activity on protein expression levels, mass spectrometry was used for proteomics analysis of HEK 293T cells when PspCas13b was co-expressed with non-targeted (NT) crRNA or targeting (T) crRNA, which targets the BCR-ABL1 mRNA breakpoint. There was no evidence of off-target protein degradation by T crRNA compared to the non-targeting crRNA. The only significantly repressed proteins were the target BCR-ABL1 p190 (81% reduction) and eGFP (89% reduction) that are encoded on the same mRNA expression construct and encoded mRNA. Catalytic dead Cas13 (dCas13) and crRNA only were used as negative control.

2024-05-20 | PXD051089 | Pride

Assessing the reliability of spike-in normalization for analyses of single-cell RNA sequencing data

Project description:This study aims to assess the reliability of spike-in normalization for analyzing single-cell RNA sequencing data. This is done by performing mixture experiments where two different sets of spike-in RNA (ERCC and SIRV) are added separately to a single mouse cell (416B or trophoblast stem cells (TSCs)), followed by generation of sequencing libraries using a modified version of the Smart-seq2 protocol. The aim is to measure the variance of the log-ratio of the total counts between the two spike-in sets. This will quantify how precisely the spike-in RNA was added to each well. As a control, addition was also performed with a premixed solution of both spike-ins, to quantify the variability in the log-ratios due to the experimental protocol. The same data can also be used to measure the well-to-well variability in the differences in behaviour between the two spike-in sets. The data contain four batches of libraries (block), using different batches of cells that were processed and sequenced separately. Each batch contains libraries with all three types of spike-in addition (ERCC+SIRV, SIRV+ERCC or Premixed). The 416B cells also contain a CBFB-MYH11 oncogene, which is expressed in half of the cells (Induced) and silent in the other half (Control).

2017-05-31 | E-MTAB-5522 | biostudies-arrayexpress

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