Journal Article

Cytosine base editing systems with minimized off-target effect and molecular size

Kondo, Akihiko, Yoshioka, Shin, Li, Ang, Nishida, Keiji, Mitsunobu, Hitoshi, Suzuki, Takahisa

2022

Overview

Cytosine base editing enables the installation of specific point mutations without double-strand breaks in DNA and is advantageous for various applications such as gene therapy, but further reduction of off-target risk and development of efficient delivery methods are desired. Here we show structure-based rational engineering of the cytosine base editing system Target-AID to minimize its off-target effect and molecular size. By intensive and careful truncation, DNA-binding domain of its deaminase PmCDA1 is eliminated and additional mutations are introduced to restore enzyme function. The resulting tCDA1EQ is effective in N-terminal fusion (AID-2S) or inlaid architecture (AID-3S) with Cas9, showing minimized RNA-mediated editing and gRNA-dependent/independent DNA off-targets, as assessed in human cells. Combining with the smaller Cas9 ortholog system (SaCas9), a cytosine base editing system is created that is within the size limit of AAV vector. Base editing is promising for gene therapy, but in vivo delivery has been limiting. Here the authors perform structure-based rational engineering of the cytosine base editing system Target-AID to minimise off-target effects and decrease its size.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 13 / 13/109 / 42/70 / 631/208/4041/3196 / 631/326/596/1278 / 631/61/338/552 / CRISPR-Associated Protein 9 - genetics / CRISPR-Associated Protein 9 - metabolism / CRISPR-Cas Systems - genetics / Cytosine / Deoxyribonucleic acid / DNA / DNA - genetics / DNA damage / Editing / Gene Editing - methods / Gene therapy / gRNA / Humanities and Social Sciences / Humans / multidisciplinary / Mutation / Recovery of function / RNA editing / RNA, Guide, CRISPR-Cas Systems / Science / Science (multidisciplinary)