Bamidele, NathanZhang, HanDong, XiaolongCheng, HaoyangGaston, NicholasFeinzig, HaileyCao, HanbingKelly, KarenWatts, Jonathan KXie, JunGao, GuangpingSontheimer, Erik J2024-04-022024-04-022024-02-17Bamidele N, Zhang H, Dong X, Cheng H, Gaston N, Feinzig H, Cao H, Kelly K, Watts JK, Xie J, Gao G, Sontheimer EJ. 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. PMID: 38368418; PMCID: PMC10874451.2041-172310.1038/s41467-024-45763-538368418https://hdl.handle.net/20.500.14038/53258Nme2Cas9 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.enOpen Access: This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/ licenses/by/4.0/. © The Author(s) 2024Attribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/CRISPR-Cas9 genome editingMolecular medicineTargeted gene repairJournal ArticleNature communications