Identification of collateral RNA cleavage mediated by Type VI CRISPR-Cas system from Leptotrichia shahiii
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ABSTRACT:
PROVIDER: PRJNA758987 | ENA |
REPOSITORIES: ENA
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Project description:We investigated the in vivo and in vitro specificity of collateral RNA degradation mediated by LshCas13a protein encoded by Type VI CRISPR-Cas system derived from L. shahii. In our experimental system, we used Escherichia coli strain harboring Type VI spacer matching inducible transcript ("targeting" cells) or strain that does not encode spacers matching any E. coli transcripts ("nontargeting" cells). After the induction of the target transcription, cells were collected, total RNA samples were extracted from the cells and subjected to high throughput RNA sequencing with a specific protocol allowing to capture RNA degradation products. To investigate the influence of RNase toxins encoded in E. coli genome on the observed RNA degradation pattern, we repeated the described experiment on E. coli strain lacking ten known ribonuclease-encoding toxin-antitoxin loci. To investigate in vitro specificity of LshCas13a protein, we analyzed products of the degradation on total E. coli RNA by purified LshCas13a supplemented with crRNA and targeted or nontargeted transcripts. The obtained samples were processed as it was described above. All experiments were independently performed in triplicate. To detect transcript degradation sites, the obtained read pairs were filtered using trimmomatic tool and then mapped onto reference sequences using bowtie2. Next, for each nucleotide position of each strand of reference sequences the number of 5’ ends of aligned fragments were counted producing corresponding tables. The differences between the numbers of mapped 5’ ends in targeting and nontargeting samples were analyzed using edgeR package. Using this data, we identified target preferences for collateral RNA degradation by LshCas13a effectors.
2022-08-31 | GSE183061 | GEO
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
Project description:Transcriptional response to collateral RNA cleavage by LbuCas13a
| PRJNA911286 | ENA
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.
2021-12-23 | GSE191329 | GEO
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.
2024-02-28 | GSE260500 | GEO