DNA capture by a CRISPR-Cas9-guided adenine base editor.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• CRISPR-Cas-guided base editors enable precise DNA modifications, converting A•T to G•C or C•G to T•A.
• Adenine base editors (ABEs) are crucial tools for targeted genome editing.
• Understanding the molecular mechanisms of ABEs is essential for improving their efficiency and specificity.

Purpose of the Study:

• To elucidate the molecular basis for the enhanced DNA adenosine deamination activity of adenine base editors (ABEs).
• To determine the structural features contributing to the high efficiency of the ABE8e variant.

Main Methods:

• Determined a 3.2-angstrom resolution cryo-electron microscopy (cryo-EM) structure of ABE8e in a substrate-bound state.
• Analyzed kinetic data to quantify deamination rates.
• Investigated the conformational changes of DNA within the CRISPR-Cas9 R-loop complex.

Main Results:

• The cryo-EM structure revealed the deaminase domain of ABE8e engaging DNA within the CRISPR-Cas9 R-loop.
• Mutations in ABE8e stabilize DNA substrates in a constrained, transfer RNA-like conformation, increasing deamination rates up to ~1100-fold compared to earlier ABEs.
• Accelerated deamination suggests a transient DNA melting mechanism during CRISPR-Cas9 surveillance.

Conclusions:

• The structural and kinetic data explain the enhanced base-editing outcomes mediated by ABE8e.
• The findings provide a molecular rationale for ABE8e's superior performance.
• This study informs the rational design of next-generation base editors for precision genome editing.