CircRNA sequence authenticity in live cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE221086 | GEO | 2023/12/31
REPOSITORIES: GEO
Similar Datasets
Project description:Base editors (BEs) shed new light on correcting disease-related T-to-C mutations. However, current rat APOBEC1-based BEs are less efficient in editing cytosines in highly-methylated regions or in GpC context. By screening a variety of APOBEC/AID deaminases, we showed that human APOBEC3A-conjugated BE and its engineered forms can mediate efficient C-to-T base editing in all examined contexts, including regions with high-methylation levels and GpC dinucleotides, which extends base editing scope.
2018-08-20 | GSE114999 | GEO
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 | GSE129889 | GEO
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
Project description:Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the selected genomic locus, and is limited to a window within the CRISPR-Cas R-loop where single stranded (ss)DNA is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility, and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with an adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABE) with PAM-proximally shifted editing windows. HNHx-ABEs are substantially reduced in size, and expand the targeting scope of base editors. Our finding that the HNH domain is replaceable could moreover benefit future protein engineering efforts, where Cas9 operates together with other enzyme domains.
2020-11-12 | GSE161293 | GEO
Project description:Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the selected genomic locus, and is limited to a window within the CRISPR-Cas R-loop where single stranded (ss)DNA is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility, and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with an adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABE) with PAM-proximally shifted editing windows. HNHx-ABEs are substantially reduced in size, and expand the targeting scope of base editors. Our finding that the HNH domain is replaceable could moreover benefit future protein engineering efforts, where Cas9 operates together with other enzyme domains.
2020-11-12 | GSE161380 | GEO
Project description:Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the selected genomic locus, and is limited to a window within the CRISPR-Cas R-loop where single stranded (ss)DNA is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility, and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with an adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABE) with PAM-proximally shifted editing windows. HNHx-ABEs are substantially reduced in size, and expand the targeting scope of base editors. Our finding that the HNH domain is replaceable could moreover benefit future protein engineering efforts, where Cas9 operates together with other enzyme domains.
2020-11-12 | GSE161379 | GEO
Project description:We conceived that the availability of base editors and the possible presence of suboptimal Kozak sequences in the human genome could provide an elegant approach to modulate translational efficiency for the molecular compensation of some haploinsufficient disorders. Moreover, such an approach would be independent of the type of alteration inactivating the defective allele and, therefore, highly suitable for gene therapy protocols. To find base conversions upregulating translational efficiency, we systematically screened 5261 (4621 unique) variants of Kozak sequences for translational strength. We designed these variants in the main AUG context of 230 previously annotated haploinsufficient genes. Analysis of our library of Kozak variants, bearing the conversions that base editors can reproduce, proved that weak Kozak sequences are present at about a 20% frequency in our gene sample. We validated the variants of five genes and base edited one of them in a cell model, providing proof of principle of the approach, which we called BOOST (Base editing cOrrection of haplOinSufficiency by Translational enhancement).
2022-09-13 | GSE210035 | GEO
Project description:The genome-wide identification, tissue-specificity and functional implications of Apobec-1 mediated C-to-U RNA editing remains incomplete. Deep sequencing, data filtering and validation from wild-type and Apobec-1 deficient mice revealed 56 novel editing sites in 54 intestinal mRNAs and 22 novel sites in 17 liver mRNAs (74-81% true-positive), all within 3' untranslated regions. Eleven of 17 liver RNAs shared editing sites with intestinal RNAs, while 6 sites were unique to liver. Changes in RNA editing led to corresponding changes in intestinal mRNA and protein levels in 11 genes. We found distinctive polysome profiles for several editing targets and demonstrated nuclear but not cytoplasmic editing of novel exonic sites in intestinal (but not hepatic) apoB RNA. RNA editing was validated using cell-free extracts from wild-type but not Apobec-1 deficient mice. These studies define selective, tissue-specific targets of Apobec-1 dependent RNA editing and show the functional consequences of editing are both transcript- and tissue-specific. Examination of C-to-U RNA editing in mouse liver and intestine
2014-05-23 | E-GEOD-57910 | biostudies-arrayexpress
Project description:The AID (activation induced deaminase)/APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) family of proteins are zinc-dependent cytidine deaminases active on polynucleotides and involved in RNA editing, DNA demethylation or DNA editing. The enzymatic activity, substrate or physiological target(s) of APOBEC2, a member of the AID/APOBEC, remain elusive. Here, we combined next generation sequencing (NGS) techniques with state-of-the-art molecular biology to comprehensively examine the physiological effects of APOBEC2 on the transcriptome and methylome using C2C12 myoblasts differentiating in culture. We also examined APOBEC2’s genome wide-binding specificity. Using RNA sequencing (RNA-seq) by polyA capture, we detected no evidence that APOBEC2 is an RNA editor. In the same system, enhanced reduced representation bisulfite sequencing (eRRBS) data did not support the proposed role of APOBEC2 as a 5-methyl-C (5mC) deaminase. However, chromatin immunoprecipitation sequencing (ChIP-Seq) did reveal specific locations of genomic occupancy of APOBEC2 with a specific motif preference. Combining biochemical, ChIP-Seq and RNA-Seq gene expression analyses we demonstrate that APOBEC2 acts as a negative regulator of gene expression in muscle cells. Our data support a model of APOBEC2 acting as a chromatin-binding factor that leads to inhibition of transcription of genes involved in cell cycle regulation during C2C12 differentiation.
2021-07-26 | GSE117730 | GEO
Project description:The AID (activation induced deaminase)/APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) family of proteins are zinc-dependent cytidine deaminases active on polynucleotides and involved in RNA editing, DNA demethylation or DNA editing. The enzymatic activity, substrate or physiological target(s) of APOBEC2, a member of the AID/APOBEC, remain elusive. Here, we combined next generation sequencing (NGS) techniques with state-of-the-art molecular biology to comprehensively examine the physiological effects of APOBEC2 on the transcriptome and methylome using C2C12 myoblasts differentiating in culture. We also examined APOBEC2’s genome wide-binding specificity. Using RNA sequencing (RNA-seq) by polyA capture, we detected no evidence that APOBEC2 is an RNA editor. In the same system, enhanced reduced representation bisulfite sequencing (eRRBS) data did not support the proposed role of APOBEC2 as a 5-methyl-C (5mC) deaminase. However, chromatin immunoprecipitation sequencing (ChIP-Seq) did reveal specific locations of genomic occupancy of APOBEC2 with a specific motif preference. Combining biochemical, ChIP-Seq and RNA-Seq gene expression analyses we demonstrate that APOBEC2 acts as a negative regulator of gene expression in muscle cells. Our data support a model of APOBEC2 acting as a chromatin-binding factor that leads to inhibition of transcription of genes involved in cell cycle regulation during C2C12 differentiation.
2021-07-26 | GSE117729 | GEO