Cas9 cleavage assay for pre-screening of sgRNAs using nicking triggered isothermal amplification

Chemical Science
Kaixiang ZhangJinghong Li

Abstract

CRISPR/Cas9 is a highly efficient genome engineering tool, yet its off-target effects and sequence-dependent cleavage activity across different sgRNAs remain major concerns for its application. Here, we propose a nicking triggered exponential amplification reaction (NTEXPAR), a fast and sensitive in vitro method, to detect the double strand DNA cleaved by down to 10 pM Cas9 with a linear range of 100 pM to 20 nM. With this newly developed amplification method, Cas9 cleavage activity can be quantified in 40 min and the optimal sgRNA design for specific target sequence can be successfully determined. Using the pre-screened sgRNA, we are able to distinguish single nucleotide mismatch in a gene silencing experiment. This fluorescence based isothermal assay provides a versatile tool for the pre-screening of sgRNAs to achieve highly specific and highly efficient CRISPR/Cas9 genome editing.

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Citations

Feb 29, 2020·Chemical Society Reviews·Kaixiang ZhangJinghong Li
Jul 8, 2020·Chemical Communications : Chem Comm·Shaohua GongBo Tang
Oct 19, 2017·Chemical Communications : Chem Comm·Jing WangFuan Wang
Oct 6, 2020·Plant Direct·Heinrich BenteMattia Donà
May 15, 2018·Analytical Chemistry·Kyle J SeamonBrooke Harmon
Aug 21, 2018·Journal of the American Chemical Society·Kaixiang ZhangJinghong Li
Feb 28, 2019·Analytical Chemistry·Yue LiJinghong Li
Mar 30, 2017·Accounts of Chemical Research·Ruijie DengJinghong Li
Jul 26, 2018·Langmuir : the ACS Journal of Surfaces and Colloids·Min PanFuan Wang
Jul 26, 2019·Analytical Chemistry·Jianyu HuYi Lv

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Methods Mentioned

BETA
cleavage assay
electrophoresis
PCR
fluorescence imaging
in vitro transcription
fluorescence microscopy

Software Mentioned

EXPAR
NTEXPAR

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