Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope

BMC Biol. 2023 Nov 9;21(1):250. doi: 10.1186/s12915-023-01754-4.

Abstract

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.

Keywords: Adenine base editors; Inlaid domain; Nme2ABE8e-797; Nme2ABE8e-797Smu; Nme2ABE8e-797−C.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenine / chemistry
  • Animals
  • CRISPR-Associated Protein 9* / genetics
  • CRISPR-Cas Systems*
  • DNA / genetics
  • Gene Editing / methods
  • Humans
  • Mammals / genetics
  • Mice

Substances

  • CRISPR-Associated Protein 9
  • Adenine
  • DNA