Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1220701 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR32268600.fastq.gz | Fastqsanger.gz | |||
| SRR32268700.fastq.gz | Fastqsanger.gz | |||
| SRR32268800.fastq.gz | Fastqsanger.gz | |||
| SRR32268601.fastq.gz | Fastqsanger.gz | |||
| SRR32268701.fastq.gz | Fastqsanger.gz |
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Project description:The most common form of genetic heart disease is hypertrophic cardiomyopathy (HCM), which is caused by mutations in cardiac sarcomeric genes and leads to abnormal heart muscle thickening. Complications of HCM include heart failure, arrhythmia, and sudden cardiac death. The dominant-negative c.1208 G>A (p.R403Q) mutation in b-myosin (MYH7) is a common and well-studied mutation that leads to increased cardiac contractility and HCM onset. Here we identify an adenine base editor (ABE) and single-guide RNA system that can efficiently correct this human pathogenic mutation with minimal off-target and bystander editing. We show that delivery of base editing components rescues pathological manifestations of HCM in iPSC-cardiomyocytes derived from HCM patients and in a humanized mouse model of HCM. Our findings demonstrate the use of base editing to treat inherited cardiac diseases and prompt the further development of ABE-based therapies to correct a variety of monogenic mutations causing cardiac disease.
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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. This SuperSeries is composed of the SubSeries listed below.
2019-03-31 | GSE121668 | GEO
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
Project description:Even the latest generation of base editor (BE3) causes unwanted indels and non-C-to-T substitutions, compromising the fidelity of base editing outcome. Here we report a enhanced base editing system. The enhanced base edting system decreased the formation of unintended indels and C-to-A/C-to-G substitutions, and increased the frequency of desired C-to-T substitution, thereby improving both the accuracy and efficiency of base editing.
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Project description:Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show Efficacy of cytosine and adenine base editors in modelingCTNNB1hot-spot mutations in hepatocyte organoids. Next, we use C>T base editors to insert nonsense mutations inPTENin endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug screening assays on organoids harboring eitherPTENorPTENandPIK3CAmutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C>T and A>G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.
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Project description:Engineering of adenine base editor with improved editing activity across NNN PAMs
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Project description:In this study, we set out to reprogram deaminase context specificity to pinpoint editing. We identified multiple nucleic acid-recognition hotspots in the E. coli tRNA-specific adenosine deaminase (TadA). Strategically sampling these recognition hotspots, we first accessed multipotency for C in TadA and subsequently eliminate its A-deamination activity. We further reprogrammed TadAC context specificity through 16 evolution campaigns, each aimed at a defined NCN context, and isolated hundreds of thousands of context-specific cytosine deaminases. Our panel of 16 NCN-specific deaminases covers the full spectrum of all possible minus1 and plus 1 contexts for a target C, offering on demand deaminase choices for editor customization. Our context-specific CBEs corrected 5,866 of 7,196 disease-associated T:A-to-C:G transitions documented by ClinVar with higher accuracy than existing CBEs, often achieving selective editing of a single cytosine out of multiple cytosines in the protospacer without compromising editing potency. We also showcased the application of context-specific base editing for modeling disease-associated C:G-to-T:A transitions using two cancer driver mutations, KRASG12D and TP53R248Q, each demanding selective editing of one cytosine in two consecutive cytosines (ACC and CCG). These context-specific editors, as expected, showed tightly controlled off-target profiles by rejecting most cytosines at potential off-target sites. Bystander-free, single-nucleobase editing, as enabled by reprogramming deaminase context specificity, complements our current editor portfolio and unlocks new potential in base editing.
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Project description:Adenine and cytosine base editors (ABEs and CBEs) represent a new genome editing technology that allows the programmable installation of A-to-G or C-to-T alterations on DNA. We engineered Streptococcus pyogenes Cas9-based adenine and cytosine base editor (SpACE) that enables efficient simultaneous introduction of A-to-G and C-to-T substitutions in the same base editing window on DNA.
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