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Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope [DNA-seq]

ABSTRACT: Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope [DNA-seq]

PROVIDER: PRJNA677990 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR13050300_1.fastq.gz	Fastqsanger.gz
	SRR13050300_2.fastq.gz	Fastqsanger.gz
	SRR13050400_1.fastq.gz	Fastqsanger.gz
	SRR13050400_2.fastq.gz	Fastqsanger.gz
	SRR13050301_1.fastq.gz	Fastqsanger.gz

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Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

Project description:Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

| PRJNA677983 | ENA

Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

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

Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope [DNA-seq]

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Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope [RNA-seq]

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Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope [RNA-seq]

Project description:Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope [RNA-seq]

| PRJNA677989 | ENA

Replacing the <i>Sp</i>Cas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope.

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 target locus and is limited to a window within the CRISPR-Cas R-loop, where single-stranded DNA (ssDNA) 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 a monomeric or heterodimeric adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABEs) with PAM-proximally shifted editing windows. This work expands the targeting scope of base editors and provides base editor variants that are substantially smaller. It moreover informs of potential future directions in Cas9 protein engineering, where the HNH domain could be replaced by other enzymes that act on ssDNA.

| S-EPMC8479293 | biostudies-literature

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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MESO-SCOPE, SCOPE cruise July 2017

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Detecting non-canonically-targeted SpCas9 generated DSBs in human cells by LAM-HTGTS

Project description:We identified robust cleavage by non-canonically-targeted SpCas9 that target protospacer with a few bases from the canonical NGG PAM in biochemistry assay. To determine whether this spaced SpCas9 cleavage exists in human cells, we employed high-throughput genome-wide translocation sequencing (LAM-HTGTS) using SpCas9 with three independent canonical sgRNAs served as bait (bait-A, bait-L and bait-D) to compared the cleavage activities of canonically-targeted and non-canonically-targeted SpCas9 in eight locus (RAG1B-D, RAG1K-O) in chromosome 11. We found that almost all 1bp-target shifted SpCas9 generated substantial DSBs as the canonical controls. While detected less frequently, 2bp-targeted shifted SpCas9 generated significant amounts of DSBs in RAG1D and RAG1L locus. These findings underscore the spaced PAM-mediated DSBs occurred in living cells.

2022-02-28 | GSE192459 | GEO

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Project description:In this study, we used a surrogate lentivirus library to capture CRISPR editing outcome in HEK cells. The dataset include quantification of indel frequencies for SpCas9 gRNAs in 12,000 surrogate sites. After filtering low quality sites, the high quality SpCas9 gRNA activities from a total of 10592 sites have been used to develop an improved deep learning-based prediction model CRISPRon (https://rth.dk/resources/crispr/.).

2021-05-03 | GSE173708 | GEO

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