Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells

Area of Science:

• Molecular Biology
• Genomics
• Structural Biology

Background:

• Conventional genome editing relies on DNA double-strand breaks (DSBs), leading to unpredictable outcomes for large DNA insertions.
• CRISPR-associated transposases (CASTs) offer a novel DSB-free approach for DNA integration, but efficiency has been limited.

Purpose of the Study:

• To elucidate the structural basis of DNA recognition by the PseCAST QCascade complex.
• To engineer improved CAST variants for enhanced DNA integration efficiency and specificity in human cells.

Main Methods:

• Single-particle cryogenic electron microscopy (cryo-EM) to determine the structure of the PseCAST QCascade complex.
• Library screening and rational mutagenesis to identify and engineer CAST variants.
• Development of hybrid CASTs by combining orthogonal DNA-binding and integration modules.

Main Results:

• Cryo-EM revealed subtype-specific DNA interactions and RNA-DNA heteroduplex features of the PseCAST QCascade complex.
• Engineered CAST variants demonstrated significantly increased DNA integration efficiencies and altered protospacer adjacent motif (PAM) specificities.
• Hybrid CASTs were successfully constructed, integrating distinct DNA-binding and catalytic modules.

Conclusions:

• Structural insights into type I-F CASTs provide a foundation for understanding and engineering RNA-guided transposase systems.
• The developed strategies offer diverse approaches to optimize CASTs for precise and efficient genome editing applications.