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Comprehensive evaluation of adenine base editors induced mutations at genomic and transcriptional level

ABSTRACT: Comprehensive evaluation of adenine base editors induced mutations at genomic and transcriptional level

PROVIDER: PRJNA769274 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR16230500_1.fastq.gz	Fastqsanger.gz
	SRR16230500_2.fastq.gz	Fastqsanger.gz
	SRR16230501_1.fastq.gz	Fastqsanger.gz
	SRR16230501_2.fastq.gz	Fastqsanger.gz
	SRR16230502_1.fastq.gz	Fastqsanger.gz

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Comprehensive evaluation of adenine base editors induced mutations at genomic and transcriptional level [WGS]

Project description:Adenine base editors (ABEs) with wide CRISPR compatibility and high activity improves the editing efficiency and arouses the off-target challenges as well. Here, we carried out a comprehensive evaluation of ABE8e and ABE9 induced DNA and RNA mutations in model organism rice. The whole-genome sequencing analysis on plants with rBE46b (SpCas9n-TadA8e), rBE49b (SpCas9n-TadA9), rBE50 (SpCas9n-NG-TadA8e), rBE53 (SpCas9n-NG-TadA9) reveals that the ABEs with TadA9 lead to a higher number of off-target A>G SNVs and ABEs with SpCas9n-NG lead to a higher total number of off-target SNVs. The analysis of T-DNAs (ABEs carrier) disclosed that the on-target mutations could happen before T-DNA integration to plant genomes as well as after T-DNA integration to plant genomes, while ABEs integrated into plant genomes lead to more A>G SNVs. We also detected off-target A>G RNA mutations in plants with higher expression of ABEs but not in plants with lower expression of ABEs. The off-target A>G RNA mutations tend to cluster while off-target A>G DNA mutations cluster in a very rare manner. The findings that CRISPRs, TadA variants, T-DNA integration, and ABE expression contribute ABEs’ specificity provide alternative ways to increase the specificity of ABEs.

2022-01-01 | GSE185495 | GEO

Comprehensive evaluation of adenine base editors induced mutations at genomic and transcriptional level [transcriptome]

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Comprehensive evaluation of adenine base editors induced mutations at genomic and transcriptional level [transcriptome]

Project description:Comprehensive evaluation of adenine base editors induced mutations at genomic and transcriptional level [transcriptome]

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Comprehensive evaluation of adenine base editors induced mutations at genomic and transcriptional level [WGS]

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Targeted exon skipping with AAV-mediated split adenine base editors.

Project description:Techniques for exclusion of exons from mature transcripts have been applied as gene therapies for treating many different diseases. Since exon skipping has been traditionally accomplished using technologies that have a transient effect, it is particularly important to develop new techniques that enable permanent exon skipping. We have recently demonstrated that this can be accomplished using cytidine base editors for permanently disabling the splice acceptor of target exons. We now demonstrate the application of adenine-deaminase base editors to disrupt the conserved adenosine within splice acceptor sites for programmable exon skipping. We also demonstrate that by altering the amino acid sequence of the linker between the adenosine deaminase domain and the Cas9 nickase or by coupling the adenine base editor with a uracil glycosylase inhibitor, the DNA editing efficiency and exon skipping rates improve significantly. Finally, we developed a split base editor architecture compatible with adeno-associated viral packaging. Collectively, these results represent significant progress towards permanent in vivo exon skipping through base editing and, ultimately, a new modality of gene therapy for the treatment of genetic diseases.

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Transcriptome-wide effects of cytosine and adenine deaminases on gene expression induced by base editors

Project description:We used RNA-sequencing data to explore the transcriptome-wide effects of cytosine and adenine deaminases on gene expression induced by base editors.

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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.

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Precise adenine base editors that exhibit minimized cytosine catalysis

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Comprehensive evaluation of adenine base editors induced mutations at genomic and transcriptional level

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

2022-01-01 | GSE185497 | GEO