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 and Erik J. Sontheimer ()
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Nathan Bamidele: RNA Therapeutics Institute, University of Massachusetts Chan Medical School
Han Zhang: RNA Therapeutics Institute, University of Massachusetts Chan Medical School
Xiaolong Dong: Tessera Therapeutics
Haoyang Cheng: RNA Therapeutics Institute, University of Massachusetts Chan Medical School
Nicholas Gaston: RNA Therapeutics Institute, University of Massachusetts Chan Medical School
Hailey Feinzig: RNA Therapeutics Institute, University of Massachusetts Chan Medical School
Hanbing Cao: RNA Therapeutics Institute, University of Massachusetts Chan Medical School
Karen Kelly: RNA Therapeutics Institute, University of Massachusetts Chan Medical School
Jonathan K. Watts: RNA Therapeutics Institute, University of Massachusetts Chan Medical School
Jun Xie: Horae Gene Therapy Center, University of Massachusetts Chan Medical School
Guangping Gao: Horae Gene Therapy Center, University of Massachusetts Chan Medical School
Erik J. Sontheimer: RNA Therapeutics Institute, University of Massachusetts Chan Medical School

Nature Communications, 2024, vol. 15, issue 1, 1-13

Abstract: 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.

Date: 2024
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DOI: 10.1038/s41467-024-45763-5

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