Computational reprogramming of homing endonuclease specificity at multiple adjacent base pairs

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Site-specific homing endonucleases facilitate gene conversion through homologous recombination.
• Reprogramming endonuclease specificity enables targeted gene correction at specific DNA sites.

Purpose of the Study:

• To computationally design endonucleases with altered cleavage specificities for multiple base pair substitutions.
• To investigate the efficacy of concerted versus modular design approaches for reprogramming endonuclease specificity.
• To explore the use of structural information in iterative design for large-scale specificity switching.

Main Methods:

• Utilized computational protein design to modify the cleavage specificity of the I-MsoI endonuclease.
• Employed a concerted design strategy targeting three simultaneous base pair substitutions.
• Incorporated crystal structure analysis of designed protein-DNA complexes for iterative design refinement.
• Developed an endonuclease capable of cleaving DNA sites with four contiguous base pair substitutions.

Main Results:

• Achieved an endonuclease with activity and specificity comparable to the wild-type enzyme after redesigning for three base pair substitutions.
• Demonstrated that concerted design approaches are more effective than modular ones for reprogramming specificity.
• Identified significant, unanticipated DNA conformational changes through structural analysis.
• Successfully designed an endonuclease for a four base pair substitution site.

Conclusions:

• Computational design enables the reprogramming of endonuclease specificity for multiple, concerted base pair substitutions.
• Iterative design cycles combining computational modeling and structural determination are crucial for large-scale specificity alterations.
• This approach offers a powerful strategy for engineering custom endonucleases for precise gene editing applications.