S. aureus BE3.9max C-terminal Fragment

C-terminal fragment of optimized SaCas9 base editor split via Npu intein at Cys535. Pairs with N-terminal for high-efficiency editing.

Length: 1000 bp (333 aa)

Type: Cytosine Base Editor

Conversion: C-to-T

Origin: S. aureus Cas9 D10A nickase (amino acids 535-1053) with Npu intein C-terminal domain

Characteristics

Contains C-terminal portion of SaCas9 D10A (residues 535-1053) including PAM recognition and DNA binding domains for NNGRRT PAM. Split site at Cys535 enables Npu intein trans-splicing. BE3.9max optimizations (enhanced NLS, codon usage) provide improved efficiency. Reference AAV construct expresses from Cbh promoter.

Applications

High-efficiency compact dual-delivery cytosine base editing. Successfully demonstrated in brain, liver, retina, heart, and muscle. Can be adapted to other delivery platforms when combined with N-terminal fragment. Enables C-to-T editing at loci requiring compact editors.

Limitations

Non-functional without N-terminal pair. Large total coding sequence (~5.6 kb across both AAVs) approaches packaging limits. NNGRRT PAM restricts targeting compared to SpCas9. Must be co-delivered with split-sabe39max-nt for C-to-T editing activity.

Literature References

  1. Komor et al. (2016). Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature - Komor 2016 BE3
  2. Komor et al. (2017). Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv - Komor 2017 BE4
  3. Levy et al. (2020). Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. Nat Biomed Eng - Levy 2020 BE3.9max
  4. Koblan et al. (2018). Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol - Koblan 2018 BE4max