Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity

Analytical Chemistry
Kyle J SeamonBrooke Harmon

Abstract

The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electroph...Continue Reading

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Citations

Mar 29, 2020·Nature Microbiology·Caroline MahendraJoseph Bondy-Denomy
Oct 1, 2019·Analytical Methods : Advancing Methods and Applications·Christopher R PhaneufChung-Yan Koh
Mar 2, 2021·Biochemical Pharmacology·Christopher L BarkauKeith T Gagnon
Jun 18, 2019·Biosensors & Bioelectronics·Robert K JohnstonJason C Harper
Feb 28, 2019·Analytical Chemistry·Yue LiJinghong Li
Jul 26, 2019·Analytical Chemistry·Jianyu HuYi Lv

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