Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Discovery and evaluation of CRISPR/Cas-mediated systems for site-specific RNA editing

ABSTRACT: In this study we report a comparative, transcriptome-wide analysis of CRISPR/Cas-based RNA-editing technologies in HEK293T cells. Targeted RNA editing technologies hold great process in the area of post-transcriptional genomic engineering and impermanent therapeutic correction of disease-causing variants, though off-target editing events still remain a concern. Here, we perform paired-end poly(A)+ purified RNA-seq to characterize transcriptome-wide in HEK293T cells transfected with catalytically-dead Cas9-, Cas13b, or Cas13d fused to human ADAR2 deaminase domain containing a hyperactive editing mutation (E488Q) along with either CYFIP2¬-targeting or non-targeting gRNA, in duplicate. We find that all technologies tested introduced widespread but distinct off-target mutations in the transcriptome, with the majority of edits occurring in 3’UTR and CDS regions of genes. Many of these edits, however, were in transcripts with low coverage, with the majority of editing rates being below 20%. This study is the first to characterize Cas-based RNA editing technologies in a direct manner, and reveals the relative similarity of currently available methods both in terms of on-target and off-target efficacy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE152684 | GEO | 2020/11/01

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors [ABE-max]

Project description:CRISPR-Cas base editor technology enables targeted nucleotide alterations and is being rapidly deployed for research and potential therapeutic applications. The most widely used base editors induce DNA cytosine (C) deamination with rat APOBEC1 (rAPOBEC1) enzyme, which is targeted by a linked Cas protein-guide RNA (gRNA) complex. Previous studies of cytosine base editor (CBE) specificity have identified off-target DNA edits in human cells. Here we show that a CBE with rAPOBEC1 can cause extensive transcriptome-wide RNA cytosine deamination in human cells, inducing tens of thousands of C-to-uracil (U) edits with frequencies ranging from 0.07% to 100% in 38% - 58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, 5’ UTR, and 3’ UTR mutations. We engineered two CBE variants bearing rAPOBEC1 mutations that substantially decrease the numbers of RNA edits (reductions of >390-fold and >3,800-fold) in human cells. These variants also showed more precise on-target DNA editing and, with the majority of gRNAs tested, editing efficiencies comparable to those observed with wild-type CBE. Finally, we show that recently described adenine base editors (ABEs) can also induce transcriptome-wide RNA edits. These results have important implications for the research and therapeutic uses of base editors, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.

2019-03-31 | GSE126502 | GEO

Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors

2019-03-31 | GSE121668 | GEO

MiSeq paired-end sequencing of HEK293T cells stably expressing a Cas9/gRNA-dependent editable reporter vector to test novel nucleobase-editing techniques

Project description:The goal of these experiments were to test the on-target and target-adjacent editing efficiencies of different single-nucleobase editing systems. Previous studies have shown that tethering DNA mutating enzymes to Cas9-nickase-UGI complexes results in editing of chromosomal DNA. However, these editing events encompass undesirable target-adjacent nucleobase edits. Here, we characterize a novel approach that reduces the frequency of target-adjacent editing while maintaining a high level of on-target editing.

2020-02-23 | E-MTAB-8742 | biostudies-arrayexpress

Genome editing in plants using the compact editor CasΦ

Project description:CRISPR-Cas systems have been developed as important tools for plant genome engineering. Here, we demonstrate that the hypercompact CasΦ nuclease is able to generate stably inherited gene edits in Arabidopsis, and that CasΦ guide RNAs can be expressed with either the Pol-III U6 promoter or a Pol-II promoter together with ribozyme mediated RNA processing. Using the Arabidopsis fwa epiallele we show that CasΦ displays higher editing efficiency when the target locus is not DNA methylated, suggesting that CasΦ is sensitive to chromatin environment. Importantly, two CasΦ protein variants, vCasΦ and nCasΦ, both showed much higher editing efficiency relative to the wildtype CasΦ enzyme, and yielded more offspring plants with inherited edits. Extensive genomic analysis of gene edited plants showed no off-target editing, suggesting that CasΦ is highly specific. The hypercompact size, flexible PAM and wide range of working temperatures make CasΦ an excellent addition to existing plant genome editing systems.

2022-12-14 | GSE206798 | GEO

Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

Project description:CRISPR-Cas base editors are preferred tool for genome editing as they generate desired editing without any double strand break in the genome, as double stand break is detrimental to the cells. In our study we have demonstrated the significance of base editors in editing the highly homologous HBG promoter (HBG1 and HBG2) region to introduce novel HPFH-like mutation to elevate HbF for therapeutical applications. Previous studies revealed that the base editors can cause unintended Cas-independent edits at transcriptome level. To validate off-target at RNA level, we performed a transcriptome wide analysis. The frequency of unintended edits in the HUDEP-2 stable cell lines expressing the base editors with the gRNA were not significant compared to the control. We determined the ABE mediated A to I conversion and CBE mediated C to U conversion across the base edited samples. The RNA off-target analysis was carried out with the help of REDItools v 2 tool. This data suggests that despite high on-target editing in DNA, the Cas-independent RNA off-target were not at detectable range compared to control. The differential expression of 34 selected genes which necessitate globin regulation were compared between the unedited HUDEP WT, CBE control, ABE control, and edited ABE (with gRNA 2/11) and edited CBE (with gRNA 2/11). We observed that there is no significant differential gene expression between the edited and control cells except the gamma and delta globin genes. These results suggest that base editors are preferred tools to edit highly homologous HBG promoter region to created HPFH-like mutations inducing HbF levels without causing double strand breaks, larger deletions and no significant RNA off-targets which are detrimental to the gene edited cells.

2022-01-02 | GSE192801 | GEO

CRISPR adenine and cytosine base editors with reduced RNA off-target activities [CBE]

Project description:CRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described “enhanced” A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications.

2019-07-11 | GSE129893 | GEO

CRISPR adenine and cytosine base editors with reduced RNA off-target activities [ABE]

2019-07-11 | GSE129889 | GEO

Cas-SF01, an engineered Cas12i nuclease is an efficient genome editing tool in mammalian cells and plants

Project description:The type V-I CRISPR-Cas system is becoming increasingly attractive for its potential utility in gene editing. However, natural nucleases often exhibit low efficiency, limiting their application. Here, we utilized structure-guided rational design and combinatorial protein engineering to optimize an uncharacterized Cas12i nuclease, Cas12i3. Accordingly, we developed Cas-SF01, a Cas12i3 variant that exhibits significantly improved gene-editing activity in mammalian cells and plants. Cas-SF01 displays comparable or superior editing performance compared to SpCas9 or recently engineered Cas12 nucleases. Further analysis of PAM recognition showed that Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs. Additionally, we identified an amino acid substitution, D876R, that markedly reduced the off-target effect while maintaining high on-target activity, leading to the development of Cas-SF01HiFi (high-fidelity Cas-SF01). Finally, we demonstrated that Cas-SF01 has robust gene-editing activity in both the monocot plant rice and dicot plant pepper. Our results suggest that Cas-SF01 can serve as a robust gene-editing platform with high efficiency and specificity for future genome editing applications across different organisms.

2023-11-05 | GSE236755 | GEO

Efficient and precise editing of endogenous transcripts with SNAP-tagged ADARs

Project description:Molecular tools to target RNA site‐specifically allow recoding of RNA information and processing. SNAP‐tagged deaminases, guided by a chemically stabilized guideRNA, enable the simultaneous editing of targeted adenosine to inosine in several endogenous transcripts, with high efficiency (up to 90%), high potency, sufficient duration, and high precision. We applied SNAP‐ADARs for the efficient and concurrent editing of two disease‐relevant signaling transcripts, KRAS and STAT1. We also show improved performance compared to the recently described Cas13b-ADAR.

2018-06-01 | GSE112787 | GEO

Genetic manipulation of Bacteriophage T4 utilizing the CRISPR-Cas13b system

Project description:T4 genome editing using CRISPR-Cas13b

| PRJEB81510 | ENA