Dimerization of the deaminase domain and locking interactions with Cas9 boost base editing efficiency in ABE8e

Nov 21, 2024Nucleic acids research

Pairing of the editing enzyme and its stable binding with Cas9 improve base editing efficiency in ABE8e

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Abstract

The ABE8e DNA deaminase exhibits enhanced editing efficiency due to its stable dimer formation.

Stable dimers of the ABE8e's DNA deaminase domain are formed more effectively than its predecessor.
The strength of is critical for the efficiency of DNA .
Specific interactions between ABE8e's dimer and DNA components are unique compared to ABE7.10.
Three key residues contribute to the improved functionality of ABE8e by balancing activity and stability.
Findings may inform the engineering of more effective base editors for precise genome editing.

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CRISPR-based DNA adenine base editors (ABEs) hold remarkable promises to address human genetic diseases caused by point mutations. ABEs were developed by combining CRISPR-Cas9 with a transfer RNA (tRNA) adenosine deaminase enzyme and through directed evolution, conferring the ability to deaminate DNA. However, the molecular mechanisms driving the efficient DNA in the evolved ABEs remain unresolved. Here, extensive molecular simulations and biochemical experiments reveal the biophysical basis behind the astonishing base editing efficiency of ABE8e, the most efficient ABE to date. We demonstrate that the ABE8e's DNA deaminase domain, TadA8e, forms remarkably stable dimers compared to its tRNA-deaminating progenitor and that the strength of TadA is crucial for DNA deamination. The TadA8e dimer forms robust interactions involving its R98 and R129 residues, the RuvC domain of Cas9 and the DNA. These locking interactions are exclusive to ABE8e, distinguishing it from its predecessor, ABE7.10, and are indispensable to boost DNA deamination. Additionally, we identify three critical residues that drive the evolution of ABE8e toward improved base editing by balancing the enzyme's activity and stability, reinforcing the TadA8e dimer and improving the ABE8e's functionality. These insights offer new directions to engineer superior ABEs, advancing the design of safer precision genome editing tools.

Key numbers

580-fold

Increase in DNA rate

Comparison of rates between ABE8e and ABE7.10

Number of amino acid substitutions

Substitutions introduced in the TadA8e domain during evolution

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Dimerization of the deaminase domain and locking interactions with Cas9 boost base editing efficiency in ABE8e

Abstract

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