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

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Conventional genome editing relies on DNA double-strand breaks (DSBs), leading to unpredictable outcomes.
• CRISPR-associated transposases (CASTs) offer a novel approach for DSB-free DNA integration.
• The type I-F CAST system, PseCAST, has shown potential for large DNA insertions in human cells but faces efficiency limitations.

Purpose of the Study:

• To elucidate the structural basis of target DNA recognition by the PseCAST QCascade complex.
• To engineer PseCAST variants with improved DNA integration efficiency and altered PAM specificity.
• To design hybrid CAST systems combining enhanced DNA binding and integration capabilities.

Main Methods:

• Single-particle cryogenic electron microscopy (cryo-EM) to determine the structure of the PseCAST QCascade complex.
• Target DNA library screens and site-directed mutagenesis to identify key protein-DNA interactions.
• Rational design and construction of chimeric CAST systems.

Main Results:

• Cryo-EM revealed novel subtype-specific interactions and RNA-DNA heteroduplex features in PseCAST.
• Engineered CAST variants demonstrated increased integration efficiency and modified PAM recognition.
• Structure-guided design enabled the creation of hybrid CASTs with enhanced DNA binding and integration modules.

Conclusions:

• Structural insights into PseCAST provide a foundation for understanding type I-F CAST mechanisms.
• Engineered CAST variants and hybrid systems represent significant advancements for DSB-free genome editing.
• This work offers diverse strategies for developing novel RNA-guided transposase architectures for therapeutic genome engineering.