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Cas12a base editors induce efficient and specific editing with low DNA damage response

ABSTRACT: Cas12a base editors induce efficient and specific editing with low DNA damage response

PROVIDER: PRJNA607538 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10969510_1.fastq.gz	Fastqsanger.gz
	SRR10969510_2.fastq.gz	Fastqsanger.gz
	SRR10969511_1.fastq.gz	Fastqsanger.gz
	SRR10969511_2.fastq.gz	Fastqsanger.gz
	SRR10969512_1.fastq.gz	Fastqsanger.gz

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Cas12a base editors induce efficient and specific editing with low DNA damage response (RNA sequencing)

Project description:The advent of base editors (BEs) holds a promising potential in correcting pathogenic-related point mutations to treat relevant diseases. Unexpectedly, Cas9 nickase (nCas9) derived BEs lead to DNA double-strand breaks, which can trigger unwanted cellular responses including a p53-mediated DNA damage response (DDR). Here, we showed that catalytically-dead-Cas12a (dCas12a) conjugated BEs induced no DNA break and minimally activated DDR proteins including H2AX, ATM, ATR and p53. We further developed a BEACON (Base Editing induced by human APOBEC3A and Cas12a without DNA break) system that fuses dCas12a to the engineered APOBEC3A with enhanced deamination efficiency and editing specificity. By using BEACON, efficient C-to-T editing was achieved at levels comparable to AncBE4max and only low levels of DDR and RNA off-target (OT) effects were triggered in mammalian cells. BEACON also induced in vivo base editing in mouse embryos and targeted C-to-T conversions were detected in F0 mice.

2020-05-05 | GSE144278 | GEO

Cas12a base editors induce efficient and specific editing with low DNA damage response (DNA targeted sequencing)

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Cas12a base editors induce efficient and specific editing with low DNA damage response (RNA sequencing)

Project description:Cas12a base editors induce efficient and specific editing with low DNA damage response (RNA sequencing)

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Cas12a base editors induce efficient and specific editing with low DNA damage response (DNA targeted sequencing)

Project description:Cas12a base editors induce efficient and specific editing with low DNA damage response (DNA targeted sequencing)

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Efficient C-to-T base editing in methylated region with human APOBEC3A

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.

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Cas12a base editors induce efficient and specific editing with low DNA damage response

Project description:This SuperSeries is composed of the SubSeries listed below.

2020-05-05 | GSE145552 | GEO

CRISPR-Cas12a base editors confer efficient multiplexed genome editing in rice.

Project description: Not available

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

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

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

Project description:Base Editing has been touted the most intelligent and precise application of the CRISPR platform so far, merging the simplicity of RNA-guided nucleases with deaminases that allow for the programmable generation of single base substitutions - without introduction of double-strand breaks. Even though the two-component system has been expected to cause off-target substitutions, studies involving cytosine base editors (CBEs) showed that in most cases, relatively few single base off-targets could be detected on DNA. We introduce the concept of multi-dimensional off-targeting, presenting an extensive amount of RNA cytidines being edited by DNA base editors. Epitranscriptomic off-target effects affected different cell lines and were independent of the guide RNAs used, suggesting Cas9-independent activity of the cytidine deaminase rAPOBEC1 on single-stranded RNA. With the help of protein engineering, we developed CBE variants with massively reduced inadvertent mutation of RNA that preserve and enhance DNA base editing capabilities.

2019-03-31 | GSE121667 | GEO

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