Presentation Information
[3ASBA-17-TA]Programmable DNA Processing Enzymes for Synthetic Biology: Structure-Guided Mechanistic Insight and Engineering
○Bo Xue1 (1. National University of Singapore (Singapore))
Keywords:
Enzyme engineering,Relaxase,CRISPR-Cas9,Thermostability,Genome amplification
Programmable DNA-processing enzymes are central to synthetic biology, but broader industrial use requires both robustness under process conditions and mechanistic insight that enables reliable repurposing. This talk integrates two structure-led studies that converge on a common theme: DNA programmability is constrained by conformational control and multi-domain coordination, and these properties can be revealed or engineered through structural approaches.
First, I will describe structure-guided engineering of Streptococcus pyogenes Cas9 to improve stability and activity at elevated temperatures while preserving its widely used NGG PAM targeting range. Using disulfide-bond design and cell-free screening, we identified CTD-stabilized variants with increased thermal stability and improved high-temperature cleavage performance. The lead variant retains near-wild-type PAM preference and shows reduced off-target cleavage under standard conditions, illustrating how stabilizing key structural checkpoints can enhance both operational robustness and functional specificity.
Second, I will present cryo-EM structures and supporting biochemistry of ZouA, a Streptomyces relaxase that drives site-specific amplification of a large chromosomal unit into megabase-scale tandem arrays. The full-length structures capture an unusual substrate engagement mode in which the trans-esterase and helicase domains simultaneously bind a single-stranded DNA target. These insights explain how ZouA couples recognition, cleavage, and downstream processing, and support a model for generating DNA intermediates compatible with rolling-circle amplification.
Together, these studies outline complementary routes to next-generation DNA technologies: engineered editors suited for demanding workflows and mechanistically grounded modules for programmable, large-scale genome rewriting and gene-dosage control.
First, I will describe structure-guided engineering of Streptococcus pyogenes Cas9 to improve stability and activity at elevated temperatures while preserving its widely used NGG PAM targeting range. Using disulfide-bond design and cell-free screening, we identified CTD-stabilized variants with increased thermal stability and improved high-temperature cleavage performance. The lead variant retains near-wild-type PAM preference and shows reduced off-target cleavage under standard conditions, illustrating how stabilizing key structural checkpoints can enhance both operational robustness and functional specificity.
Second, I will present cryo-EM structures and supporting biochemistry of ZouA, a Streptomyces relaxase that drives site-specific amplification of a large chromosomal unit into megabase-scale tandem arrays. The full-length structures capture an unusual substrate engagement mode in which the trans-esterase and helicase domains simultaneously bind a single-stranded DNA target. These insights explain how ZouA couples recognition, cleavage, and downstream processing, and support a model for generating DNA intermediates compatible with rolling-circle amplification.
Together, these studies outline complementary routes to next-generation DNA technologies: engineered editors suited for demanding workflows and mechanistically grounded modules for programmable, large-scale genome rewriting and gene-dosage control.
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