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Incorporating programmable DNA-binding domains enhances the efficiency and flexibility of miniature CRISPR base editors

ABSTRACT: Incorporating programmable DNA-binding domains enhances the efficiency and flexibility of miniature CRISPR base editors

PROVIDER: PRJNA1250402 | ENA |

REPOSITORIES: ENA

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Incorporating programmable DNA-binding domains enhances the efficiency and flexibility of miniature CRISPR base editors

Project description:The therapeutic application of base editors is currently limited by their large sizes, which are often beyond the packaging capabilities of adeno-associated viral (AAV) vectors. Despite recent progress in mega genome mining that has identified a diverse array of compact CRISPR proteins (e.g. Cas12f, TnpB, and IscB), the resulting miniature base editors are often exhibited reduced activities and a limited targeting scope, leaving a broad spectrum of disease-relevant genetic variants inaccessible. In this study, we have developed a platform, designated as Zinc Finger Proteins (ZFP)-enhanced miniature base editor (zmBE), which integrates programmable DNA-binding domains to enhance the efficiency and expand targeting scope of miniature base editors, including those based on Un1Cas12f1 and OgeuIscB. Utilizing protein language model (PLM) for the design of ZFPs further simplified development and optimization of zmBEs tailored to a specific target. Leveraging these methodologies, we engineered a zmBE that effectively induced the SMN2 exon 7 A6>G conversion and restored SMN2 exon 7 inclusion. Our study thus provides a versatile platform for developing miniature base editors for in vivo therapeutic applications.

2025-04-18 | GSE294498 | GEO

CRISPR-SKIP: Programmable Gene Splicing with Single Base Editors

Project description:CRISPR-SKIP: Programmable Gene Splicing with Single Base Editors

| PRJNA437650 | ENA

CRISPR-SKIP: Programmable Gene Splicing with Single Base Editors [gDNA]

Project description:CRISPR-SKIP: Programmable Gene Splicing with Single Base Editors [gDNA]

| PRJNA437654 | ENA

CRISPR-SKIP: Programmable Gene Splicing with Single Base Editors [off-targets]

Project description:CRISPR-SKIP: Programmable Gene Splicing with Single Base Editors [off-targets]

| PRJNA437655 | ENA

CRISPR-SKIP: Programmable Gene Splicing with Single Base Editors [RNA-seq]

Project description:CRISPR-SKIP: Programmable Gene Splicing with Single Base Editors [RNA-seq]

| PRJNA437656 | ENA

CRISPR-SKIP: Programmable Gene Splicing with Single Base Editors [gDNA]

Project description:CRISPR gene editing has revolutionized biomedicine and biotechnology by providing a simple means to engineer genes in vivo by introducing mutations at target sites in the genomic DNA of living cells. However, given the stochasticity of cellular DNA repair mechanisms and the potential for introducing mutations at off-target sites, technologies capable of introducing targeted changes with increased precision, such as cytidine deaminase single-base editors, are preferred. We here present a versatile method termed CRISPR-SKIP that utilizes cytidine deaminase single-base editors to program de-novo exon skipping by mutating target DNA bases within splice acceptor sites. Given its simplicity and precision, CRISPR-SKIP will be broadly applicable in gene therapy and synthetic biology.

2018-07-06 | GSE111644 | GEO

CRISPR-SKIP: Programmable Gene Splicing with Single Base Editors [RNA-seq]

2018-07-06 | GSE111643 | GEO

CRISPR-SKIP: Programmable Gene Splicing with Single Base Editors [off-targets]

2018-07-06 | GSE111645 | GEO

Protein language models-assisted optimization of a Uracil-N glycosylase variant enables programmable T-to-S base editing

Project description:Current base editors use DNA deaminases, including cytidine deaminase in cytidine base editor (CBE) or adenine deaminase in adenine base editor (ABE), to facilitate transition nucleotide substitutions. Combining CBE or ABE with glycosylase enzymes can induce limited transversion mutations. Nonetheless, a critical demand remains for base editors capable of generating alternative mutation types, such as T>G corrections. In this study, we leveraged pre-trained protein language models to optimize a uracil-N-glycosylase (UNG) variant with altered specificity for thymines (eTDG). Notably, after two rounds of testing fewer than 50 top-ranking variants, more than 50% exhibited over 1.5-fold enhancement in enzymatic activities. When eTDG was fused with nCas9, it induced programmable T-to-S (G/C) substitutions and corrected db/db diabetic mutation in mice (up to 55%). Our findings not only establish orthogonal strategies for developing novel base editors, but also demonstrate the capacities of protein language models for optimizing enzymes without extensive task-specific training data.

2024-01-23 | GSE253655 | GEO

Homo sapiens

Project description:miniature base editors

| PRJNA918720 | ENA