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Updated: Jul 11, 2025

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
Published on: May 25, 2018
Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope
Ding Zhao1, Xun Gao1, Jiale Zhou1
1Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Veterinary Medicine, Jilin University, Changchun, 130062, China.
Background:
Nme2ABE8e has been constructed and characterized as a compact, accurate adenine base editor with a less restrictive dinucleotide protospacer-adjacent motif (PAM: N4CC) but low editing efficiency at challenging loci in human cells. Here, we engineered a subset of domain-inlaid Nme2Cas9 base editors to bring the deaminase domain closer to the nontarget strand to improve editing efficiency.
Results:
Our results demonstrated that Nme2ABE8e-797 with adenine deaminase inserted between amino acids 797 and 798 has a significantly increased editing efficiency with a wide editing window ranging from 4 to 18 bases in mammalian cells, especially at the sites that were difficult to edit by Nme2ABE8e. In addition, by swapping the PAM-interacting domain of Nme2ABE8e-797 with that of SmuCas9 or introducing point mutations of eNme2-C in Nme2ABE8e-797, we created Nme2ABE8e-797Smu and Nme2ABE8e-797-C, respectively, which exhibited robust activities at a wide range of sites with N4CN PAMs in human cells. Moreover, the modified domain-inlaid Nme2ABE8e can efficiently restore or install disease-related loci in Neuro-2a cells and mice.
Conclusions:
These novel Nme2ABE8es with increased on-target DNA editing and expanded PAM compatibility will expand the base editing toolset for efficient gene modification and therapeutic applications.
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