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• W3017046408 abstract "CRISPR systems experience off-target effects that interfere with the ability to accurately perform genetic edits. While empirical models predict off-target effects in specific platforms, there is a gap for a wide-ranging mechanistic model of CRISPR systems based on evidence from the essential processes of target recognition. In this work, we present a first principles model supported by many experiments performed in vivo and in vitro. First, we observe and model the conformational changes and discrete stepped DNA unwinding events of SpCas9-CRISPR target recognition process in a cell-free transcription-translation system using truncated guide RNAs, confirming structural and FRET microscopy experiments. Second, we implement an energy landscape to describe single mismatch effects observed in vivo for spCas9 and Cas12a CRISPR systems. From the mismatch analysis results we uncover that CRISPR class 2 systems maintain their kinetic proofreading mechanism by utilizing intermittent energetic barriers and we show how to leverage the landscape to improve target specificity." @default.
• W3017046408 created "2020-04-24" @default.
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• W3017046408 date "2020-04-20" @default.
• W3017046408 modified "2023-09-27" @default.
• W3017046408 title "Proofreading mechanism of Class 2 CRISPR-Cas systems" @default.
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• W3017046408 hasPublicationYear "2020" @default.
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