Abstract Title

Computational Insights into Cas9 Conformational Activation and Specificity Enhancement

Presenter Name

Zhicheng ZUO

RAD Assignment Number

200

Abstract

Over the past a few years, the biotechnology harnessing the microbial CRISPR/Cas systems has revolutionized the field of genome editing. The RNA-guided endonuclease Cas9 from Streptococcus pyogenes (SpCas9) can be programmed with a synthetic single guide RNA (sgRNA) to induce site-specific double-stranded DNA (dsDNA) cleavage. Despite recent progresses in deciphering the Cas9 structural and functional mechanisms, the knowledge of the Cas9 HNH nuclease domain catalytic state remains sparse, and it remains elusive as to how the catalytic Mg2+ affects the HNH domain conformational transition. A deeper understanding of Cas9 conformational activation and its action mechanism is of fundamental importance for guiding the improvement of Cas9-mediated genome-editing specificity and efficiency. Herein we report a cross-validated catalytic state of the Cas9 HNH domain poised for cutting the target DNA strand by means of two distinct molecular dynamics (MD) simulation strategies. We note that the derived model has been in good agreement and rationalized by various available experiments. Moreover, we demonstrate the essential roles of Mg2+ for the cleavage-state formation and stability. Importantly, our study suggests additional promising mutation sites on Cas9 that could be exploited for rationally engineering more Cas9 variants with enhanced specificity.

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Research Area

Biochemistry

Presentation Type

Poster

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Computational Insights into Cas9 Conformational Activation and Specificity Enhancement

Over the past a few years, the biotechnology harnessing the microbial CRISPR/Cas systems has revolutionized the field of genome editing. The RNA-guided endonuclease Cas9 from Streptococcus pyogenes (SpCas9) can be programmed with a synthetic single guide RNA (sgRNA) to induce site-specific double-stranded DNA (dsDNA) cleavage. Despite recent progresses in deciphering the Cas9 structural and functional mechanisms, the knowledge of the Cas9 HNH nuclease domain catalytic state remains sparse, and it remains elusive as to how the catalytic Mg2+ affects the HNH domain conformational transition. A deeper understanding of Cas9 conformational activation and its action mechanism is of fundamental importance for guiding the improvement of Cas9-mediated genome-editing specificity and efficiency. Herein we report a cross-validated catalytic state of the Cas9 HNH domain poised for cutting the target DNA strand by means of two distinct molecular dynamics (MD) simulation strategies. We note that the derived model has been in good agreement and rationalized by various available experiments. Moreover, we demonstrate the essential roles of Mg2+ for the cleavage-state formation and stability. Importantly, our study suggests additional promising mutation sites on Cas9 that could be exploited for rationally engineering more Cas9 variants with enhanced specificity.