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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE161293 | GEO | 2020/11/12

REPOSITORIES: GEO

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

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.

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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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Project description:CRISPR-Cas base editor technology enables targeted nucleotide alterations and is being rapidly deployed for research and potential therapeutic applications. The most widely used base editors induce DNA cytosine (C) deamination with rat APOBEC1 (rAPOBEC1) enzyme, which is targeted by a linked Cas protein-guide RNA (gRNA) complex. Previous studies of cytosine base editor (CBE) specificity have identified off-target DNA edits in human cells. Here we show that a CBE with rAPOBEC1 can cause extensive transcriptome-wide RNA cytosine deamination in human cells, inducing tens of thousands of C-to-uracil (U) edits with frequencies ranging from 0.07% to 100% in 38% - 58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, 5’ UTR, and 3’ UTR mutations. We engineered two CBE variants bearing rAPOBEC1 mutations that substantially decrease the numbers of RNA edits (reductions of >390-fold and >3,800-fold) in human cells. These variants also showed more precise on-target DNA editing and, with the majority of gRNAs tested, editing efficiencies comparable to those observed with wild-type CBE. Finally, we show that recently described adenine base editors (ABEs) can also induce transcriptome-wide RNA edits. These results have important implications for the research and therapeutic uses of base editors, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms. This SuperSeries is composed of the SubSeries listed below.

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