RT Journal Article
SR Electronic
T1 Quantitative assaying of SpCas9-NG with fluorescent reporters
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.08.04.502727
DO 10.1101/2022.08.04.502727
A1 Alexandre Baccouche
A1 Kevin Montagne
A1 Nozomu Yachie
A1 Teruo Fujii
A1 Anthony Genot
YR 2022
UL http://biorxiv.org/content/early/2022/08/04/2022.08.04.502727.abstract
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.