Biology subjects

Strohkendl, I.

Publications and source records attributed to Strohkendl, I..

3 recordsLinked to original sources

bioRxiv2025

A Cryptic Binding Pocket Regulates the Metal-Dependent Activity of Cas9

Cas9 is a metal-dependent nuclease that has revolutionized gene editing across diverse cells and organisms exhibiting varying ion uptake, metabolism, and concentrations. However, how divalent metals impact its catalytic function, and consequently its editing efficiency in different cells, remains unclear. Here, extensive molecular simulations, Markov State Models, biochemical and NMR experiments, demonstrate that divalent metals - Mg2+, Ca2+, and Co2+ - promote activation of the catalytic HNH domain by binding within a dynamically forming divalent metal binding pocket (DBP) at the HNH-RuvC interface. Mutations in DBP residues disrupt HNH activation and impair the coupled catalytic activity of both nucleases, identifying this cryptic DBP as a key regulator of Cas9s metal-dependent activity. The ionic strength thereby promotes Cas9s conformational activation, while its catalytic activity is metal-specific. These findings are critical to improving the metal-dependent function of Cas9 and its use for genome editing in different cells and organisms.

biophysics↗

bioRxiv2023

Structural basis of Cas12a R-loop propagation on pathway to DNA cleavage

Cas12a is a CRISPR RNA-guided nuclease that engages target DNA through protein-DNA and RNA-DNA contacts. Initial PAM binding by Cas12a leads to formation of a 20bp R-loop between the complementary crRNA guide and target strand. Following specificity-determining R-loop formation, both DNA strands undergo RuvC-mediated cleavage. Current structures of Cas12a bound to its target only show the R-loop after formation, leaving an important gap in knowledge as to how Cas12a accommodates the extending R-loop and coordinates R-loop formation with nuclease activation. Here, we use cryo-EM to capture a series of kinetically trapped Cas12a R-loop intermediates and observe how Cas12a delivers each DNA strand into the RuvC active site. We show that Cas12a first interrogates target DNA via a 5bp seed, followed by dramatic Rec domain conformational flexibility to accommodate R-loop extension. Only during formation of the final R-loop base pairs do the Rec and BH domains engage in the majority of contacts with the R-loop. R-loop completion leads the nontarget strand to displace the RuvC lid and kink into the active site via a base stacking interaction. Following nontarget strand cleavage, we observe substantial Rec2 and Nuc domain dynamics as the TS is brought to the RuvC active site. Our kinetics-guided structural snapshots provide a comprehensive model describing Cas12a DNA targeting and highlight mechanistic differences between Cas12a and Cas9.

biochemistry↗

bioRxiv2018

Kinetic basis for DNA target specificity of CRISPR-Cas12a

Class II CRISPR-Cas nucleases are programmable via a single guide RNA, enabling genome editing applications in nearly all organisms. However, DNA cleavage at off-target sites that resemble the target sequence is a pervasive problem that remains poorly understood mechanistically. Here, we use quantitative kinetics to dissect the reaction steps of DNA targeting by Acidaminococcus sp Cas12a (also known as Cpf1). We show that Cas12a binds DNA tightly in two kinetically-separable steps. Protospacer-adjacent motif (PAM) recognition is followed by rate-limiting R-loop propagation, leading to inevitable DNA cleavage of both strands. Despite the functionally irreversible binding, Cas12a discriminates strongly against mismatches along most of the DNA target sequence, implying substantial reversibility during R-loop formation -a late transition state- and the absence of a seed region. Our results provide a quantitative underpinning for the DNA cleavage patterns measured in vivo and observations of greater reported target specificity of Cas12a than the Cas9 nuclease.

molecular biology↗