Ontology highlight
ABSTRACT:
PROVIDER: PRJNA503988 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8164460_1.fastq.gz | Fastqsanger.gz | |||
| SRR8164460_2.fastq.gz | Fastqsanger.gz | |||
| SRR8164461_1.fastq.gz | Fastqsanger.gz | |||
| SRR8164461_2.fastq.gz | Fastqsanger.gz | |||
| SRR8164462_1.fastq.gz | Fastqsanger.gz |
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Project description:We examined the off-targets of new base editing tools at RNA level
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Project description:CRISPR/Cas9-based genome editing has revolutionized experimental molecular biology and entered the clinical world for targeted gene therapy. Identifying DNA modifications occurring at CRISPR/Cas9 target sites is critical to determine efficiency and safety of editing tools. Here we show that insertions of LINE-1 (L1) retrotransposons can occur frequently at CRISPR/Cas9 editing sites. Together with PolyA-seq and an improved amplicon sequencing, we characterize more than 2,500 de novo L1 insertions at multiple CRISPR/Cas9 editing sites in HEK293T, HeLa and U2OS cells. These L1 retrotransposition events exploit CRISPR/Cas9-induced DSB formation and require L1 RT activity. Importantly, de novo L1 insertions are rare during genome editing by prime editors (PE), cytidine or adenine base editors (CBE or ABE), consistent with their reduced DSB formation. These data demonstrate that insertions of retrotransposons might be a potential outcome of CRISPR/Cas9 genome editing and provide further evidence on the safety of different CRISPR-based editing tools.
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Project description:Genome-wide knockout or knockdown screens have become powerful tools for the investigation of genotype-to-phenotype relationships. In bacteria, these screens commonly rely on transcriptional repression by dCas9, gene knockouts through Cas9 editing or random transposon mutagenesis, but depending on the technique, suffer from incomplete gene silencing, low editing efficiencies or they require massive library sizes. Here, we take a distinct approach with base editing to introduce premature stop codons or mutate start codons in Escherichia coli using a ScCas9 nickase derived base editor (ScBE3) that exhibits flexible PAM recognition. We then derive guide design rules by applying machine learning to a gene essentiality screen conducted in E. coli. For further improvement, we combined base-editing with Cas9-induced cleavage of the unedited cell fraction. The efficiency of this dual system was validated through a screen of conditionally essential E. coli genes. This improved setup that decouples the gene editing from the screening leads to more efficient guide depletion and confirmed previously published conditionally essential genes. Overall, base editing represents a useful tool for genome-wide knockout screens in bacteria and will eventually enable genome-wide knockout screens in a broader range of bacterial species to study their diverse genetics.
2024-02-05 | GSE225335 | GEO
Project description:Programmable base editing of RNA enables rewriting the genetic codes on specific sites. Current tools for specific RNA editing dependent on the assembly or recruitment of the guide RNA into an RNA/protein complex, which may cause delivery barrier and low editing efficiency. Here we report a new set of tools, RNA editing with individual RNA-binding enzyme (REWIRE), to perform precise base editing with a single engineered protein. The REWIRE system contains a human-originated programmable RNA-binding domain (PUF domain) to specifically recognize target sequence and different deaminase domains to achieve A-to-I or C-to-U editing. By utilizing this system, we have achieved editing efficiencies up to 80% in A-to-I editing and 65% in C-to-U editing, with a few non-specific editing sites in the targeted region and a low level off-target effect globally. We applied the REWIREs to correct disease-associated mutations and modify mitochondrial RNAs, and further optimized the REWIREs to improve the editing efficiency and minimize off-target effects. As a single-component base editing system originated from human proteins, REWIRE presents a precise and efficient RNA-editing platform with broad applicability in basic research and gene therapy.
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Project description:RNA base editing applies endogenous or engineered adenosine deaminases to introduce adenosine-to-inosine changes into a target RNA in a highly programmable manner. Recently, notable success was achieved for the repair of disease-causing guanosine-to-adenosine mutations by means of RNA base editing. Here, we propose that RNA base editing could be broadly applied to perturb protein function by removal of regulatory sites of post-translational modification (PTM), like phosphorylation and/or acetylation sites. We demonstrate the feasibility of PTM interference (PTMi) on more than 70 PTM sites in various signaling proteins and identify key determinants for high editing efficiency and potent down-stream effects. Specifically, we demonstrate both negative and positive regulation of the JAK/STAT pathway by PTMi. To identify potent regulatory sites for PTMi, we applied an improved version of the SNAP-ADAR tool, which achieved high editing efficiency over a broad codon scope with tight control of bystander editing. The transient nature of RNA base editing enables the fast, dose-dependent (thus partial) and reversible manipulation of PTM sites, which is a key advantage over DNA editing approaches, where genetic compensation or lethality can conceal a phenotype. In summary, PTM interference might become a valuable field of application of RNA base editing in basic biology and medicine.
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