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Oct 20 2023

Biophysics Seminar- “Structural basis for Cas9-mediated DNA interrogation and editing with 5’ truncated sgRNAs ” with Kaitlyn Kiernan

Biophysics Seminar

October 20, 2023

2:00 PM - 3:00 PM

Title: “Structural basis for Cas9-mediated DNA interrogation and editing with
5’ truncated sgRNAs ”

Presenter: Kaitlyn Kiernan, UIC Department of Biochemistry & Molecular Genetics

Abstract: The efficiency and accuracy of CRISPR-Cas9 targeting varies considerably across genomic targets and remains a persistent concern for implementing this system in cells and clinical studies. It has been shown that the use of 5’ truncated sgRNAs can reduce the rate of unwanted off-target recognition while still maintaining on-target specificity. However, it is not well-understood how reducing target complementarity enhances specificity or how truncation past 15-nt prevents full Cas9 activation without compromising ontarget binding. To better understand this mechanism, we performed cryo-EM analysis on a Cas9 complex bound to a truncated sgRNA. Here, we report two distinct conformations of the sgRNA-DNA heteroduplex with approximately 30% of the particles exhibiting a linear heteroduplex and find that the other 60% exhibit a kinked heteroduplex resembling that of a catalytically active conformation. Importantly, we find that reduced flexibility of the shorter non-target strand and persistent interactions with REC2 domain prevent full catalytic activation of the HNH and RuvC nuclease domains. Guided by this structural information, we designed Cas9 complexes enabling more efficient cleavage with 5’ truncated sgRNAs as well as exhibiting multi-turnover kinetics. Our results provide a structural basis for Cas9 targeting with 5’ truncated sgRNAs and support the development of new high-fidelity Cas9 complexes in future studies.



Physics Office

Date posted

Oct 20, 2023

Date updated

Oct 20, 2023