bioRxiv : the preprint server for biologyNov 18, 2024

Improving Nme2Cas9 and Nme2SmuCas9 Gene Editing Proteins and Base Editors

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Mar 10, 2026

Abstract

Adenine and cytosine base editors could correct approximately 30% of human pathogenic variants.

Base editors enable precise conversions of A:T-to-G:C and C:G-to-T:A base pairs.
Therapeutic use of base editors is limited by factors such as PAM availability and accuracy.
Nme2Cas9 and its derivatives have been developed to enhance editing near NCC PAMs.
Engineering efforts have yielded base editors with improved activity and vector compatibility.
Editing and specificity profiles of the enhanced variants were defined using paired guide-target libraries.

Simplified

CRISPR-Cas genome editing tools enable precise, RNA-guided modification of genomes within living cells. The most clinically advanced genome editors are Cas9 nucleases, but many nuclease technologies provide only limited control over genome editing outcomes. Adenine base editors (ABEs) and cytosine base editors (CBEs) enable precise and efficient nucleotide conversions of A:T-to-G:C and C:G-to-T:A base pairs, respectively. Therapeutic use of base editors (BEs) provides an avenue to correct approximately 30% of human pathogenic variants. Nonetheless, factors such as protospacer adjacent motif (PAM) availability, accuracy, product purity, and delivery limit the full therapeutic potential of BEs. We previously developed Nme2Cas9 and its BE derivatives, including ABEs compatible with single adeno-associated virus (AAV) vector delivery, in part to enable editing near NCC PAMs. Further engineering yielded domain-inlaid BEs with enhanced activity, as well as Nme2Cas9/SmuCas9 chimeras that target single-cytidine (NC) PAMs. Here we further enhance Nme2Cas9 and Nme2Cas9 editing effectors for improved efficiency and vector compatibility through site-directed mutagenesis and deaminase linker optimization. Finally, we define the editing and specificity profiles of the resulting variants by using paired guide-target libraries. 4 4Smu

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Improving Nme2Cas9 and Nme2SmuCas9 Gene Editing Proteins and Base Editors

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