Domain-inlaid Nme2Cas9 adenine base editors with improved activity and targeting scope

Nathan Bamidele; Han Zhang; Xiaolong Dong; Haoyang Cheng; Nicholas Gaston; Hailey Feinzig; Hanbing Cao; Karen Kelly; Jonathan K Watts; Jun Xie; Guangping Gao; Erik J Sontheimer

doi:10.1038/s41467-024-45763-5

Domain-inlaid Nme2Cas9 adenine base editors with improved activity and targeting scope

Nat Commun. 2024 Feb 17;15(1):1458. doi: 10.1038/s41467-024-45763-5.

Authors

Nathan Bamidele¹, Han Zhang¹, Xiaolong Dong², Haoyang Cheng¹, Nicholas Gaston¹, Hailey Feinzig¹, Hanbing Cao¹, Karen Kelly¹, Jonathan K Watts^{1

3

4}, Jun Xie^{5

6

7

8}, Guangping Gao^{5

6

7

8}, Erik J Sontheimer^{9

10

11}

Affiliations

¹ RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA.
² Tessera Therapeutics, Somerville, MA, 02143, USA.
³ Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA.
⁴ NeuroNexus Institute, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA.
⁵ Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA.
⁶ Viral Vector Core, University of Massachusetts Chan Medical, School, Worcester, MA, 01605, USA.
⁷ Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA.
⁸ Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA.
⁹ RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA. erik.sontheimer@umassmed.edu.
¹⁰ Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA. erik.sontheimer@umassmed.edu.
¹¹ Program in Molecular Medicine, University of Massachusetts Chan Medical School, Massachusetts, MA, 01605, USA. erik.sontheimer@umassmed.edu.

Abstract

Nme2Cas9 has been established as a genome editing platform with compact size, high accuracy, and broad targeting range, including single-AAV-deliverable adenine base editors. Here, we engineer Nme2Cas9 to further increase the activity and targeting scope of compact Nme2Cas9 base editors. We first use domain insertion to position the deaminase domain nearer the displaced DNA strand in the target-bound complex. These domain-inlaid Nme2Cas9 variants exhibit shifted editing windows and increased activity in comparison to the N-terminally fused Nme2-ABE. We next expand the editing scope by swapping the Nme2Cas9 PAM-interacting domain with that of SmuCas9, which we had previously defined as recognizing a single-cytidine PAM. We then use these enhancements to introduce therapeutically relevant edits in a variety of cell types. Finally, we validate domain-inlaid Nme2-ABEs for single-AAV delivery in vivo.

MeSH terms

Adenine* / metabolism
CRISPR-Associated Protein 9* / genetics
CRISPR-Associated Protein 9* / metabolism
CRISPR-Cas Systems
DNA / genetics
Gene Editing

Substances

CRISPR-Associated Protein 9
Adenine
DNA

Abstract

MeSH terms

Substances

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