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Compact zinc finger architecture utilizing toxin-derived cytidine deaminases for highly efficient base editing in human cells-K562 cells

ABSTRACT: Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddAtox, that can act upon double-stranded DNA. Here, we target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddAtox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.

ORGANISM(S): Homo sapiens

PROVIDER: GSE253184 | GEO | 2024/01/12

REPOSITORIES: GEO

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Compact zinc finger architecture utilizing toxin-derived cytidine deaminases for highly efficient base editing in human cells -T cells

Project description:Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddAtox, that can act upon double-stranded DNA. Here, we target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddAtox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.

2024-01-12 | GSE253182 | 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

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.

2019-07-11 | GSE129893 | GEO

CRISPR adenine and cytosine base editors with reduced RNA off-target activities [ABE]

2019-07-11 | GSE129889 | GEO

Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors [P2A-EGFP control]

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.

2019-04-01 | GSE129131 | GEO

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

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

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

2019-03-31 | GSE121665 | GEO