4.7 Article

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

Journal

MOLECULAR THERAPY-NUCLEIC ACIDS
Volume 26, Issue -, Pages 502-510

Publisher

CELL PRESS
DOI: 10.1016/j.omtn.2021.08.025

Keywords

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Funding

  1. Swiss National Science Foundation [180257]
  2. PHRT [528]

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Base editors are RNA-guided deaminases that enable specific nucleotide transitions at target sites. By omitting the Cas9-HNH nuclease domain, researchers expanded the editing window and engineered adenine base editor variants with PAM-proximally shifted editing windows. This work not only broadens the targeting scope of base editors but also suggests potential future directions in Cas9 protein engineering by replacing the HNH domain with other enzymes acting on ssDNA.
Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Casdeaminase 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.

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