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A comprehensive benchmark for multiple highly efficient base editors with maximal targeting scope

ABSTRACT: A comprehensive benchmark for multiple highly efficient base editors with maximal targeting scope

PROVIDER: PRJNA1198967 | ENA |

REPOSITORIES: ENA

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A comprehensive benchmark for multiple highly efficient base editors with maximal targeting scope

Project description:As the toolbox of base editors (BEs) expands, selecting appropriate BE and guide RNA (gRNA) to achieve optimal editing efficiency and outcome for a given target becomes challenging. Here we construct a set of 10 adenine and cytosine BEs with high activity and maximal targeting scope, and comprehensively evaluate their editing profiles and properties head-to-head with 34,040 BE-gRNA-target combinations using genomically integrated long targets and tiling gRNA strategies. A deep learning model BEEP (Base Editing Efficiency Predictor) is built for predicting the editing efficiency and outcomes. Guided by BEEP, we experimentally install 3,558 disease-associated single nucleotide variants (SNVs), including 20.1% of target sites that would not be edited due to known protospacer adjacent motif (PAM) restriction, and predicted candidate BE-gRNA-target combinations for modeling 1,752,651 ClinVar SNVs. We also identify several cancer-associated SNVs that drive the resistance to BRAF inhibitors in melanoma. These efforts benchmark the performance and illuminate the capabilities of multiple highly useful BEs for interrogating functional SNVs.

2025-02-12 | GSE284410 | GEO

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]

2020-11-12 | GSE161380 | GEO

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

2020-11-12 | GSE161379 | GEO

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

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.

2018-08-20 | GSE114999 | GEO

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

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

| PRJNA677990 | ENA

MESO-SCOPE, SCOPE cruise July 2017

Project description:MESO-SCOPE, SCOPE cruise July 2017 Raw sequence reads

| PRJNA981211 | ENA

Engineering Nme2Cas9 Adenine Base Editors with Improved Activity and Targeting Scope.

Project description:Nme2Cas9 has been established as a genome editing platform with compact size, high accuracy, and broad targeting range, including single-AAV-deliverable adenine base editors. Here, we have engineered Nme2Cas9 to further increase the activity and targeting scope of compact Nme2Cas9 base editors. We first used domain insertion to position the deaminase domain nearer the displaced DNA strand in the target-bound complex. These domain-inlaid Nme2Cas9 variants exhibited shifted editing windows and increased activity in comparison to the N-terminally fused Nme2-ABE. We next expanded the editing scope by swapping the Nme2Cas9 PAM-interacting domain with that of SmuCas9, which we had previously defined as recognizing a single-cytidine PAM. We used these enhancements to correct two common MECP2 mutations associated with Rett syndrome with little or no bystander editing. Finally, we validated domain-inlaid Nme2-ABEs for single-AAV delivery in vivo.

| S-EPMC10153126 | biostudies-literature

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