PT  - JOURNAL ARTICLE
AU  - Alexandre Baccouche
AU  - Kevin Montagne
AU  - Nozomu Yachie
AU  - Teruo Fujii
AU  - Anthony Genot
TI  - Quantitative assaying of SpCas9-NG with fluorescent reporters
AID  - 10.1101/2022.08.04.502727
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.08.04.502727
4099  - http://biorxiv.org/content/early/2022/08/04/2022.08.04.502727.short
4100  - http://biorxiv.org/content/early/2022/08/04/2022.08.04.502727.full
AB  - The Cas9 enzyme has revolutionized biology in less than a decade. Engineering Cas9 to expand its functionality has become a major research goal, yet assaying variants of Cas9 remains a laborious task that is commonly performed with gel electrophoresis. Fluorescence assays have been reported for Cas9 but their utility for assaying variants of Cas9 has not been investigated in detail. Here we use a simple fluorescent assay to resolve differences of activity between the wild type Streptococcus pyogenes Cas9 (SpCas9) and SpCas9-NG, a variant with an expanded PAM repertoire. We compare the kinetics of the two enzymes on dozens of mutated RNA guides – highlighting the benefits of fluorescence such as quantitativity, sensitivity, multiplexing, non-invasiveness and real-timeness. This validates fluorescence as a tool for engineering Cas9 and lays the groundwork for directly evolving Cas9 in microfluidic compartments.Competing Interest StatementAJG, TF and AB have filed a patent on Smart Guide RNA WO 2021/111641 A1.