Use of anti-CRISPR protein AcrIIA4 as a capture ligand for CRISPR/Cas9 detection.

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ID: 43020
2019
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Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex is an RNA-guided DNA-nuclease that is part of the bacterial adaptive immune system. CRISPR/Cas9 RNP has been adapted for targeted genome editing within cells and whole organisms with new applications vastly outpacing detection and quantification of gene-editing reagents. Detection of the CRISPR/Cas9 RNP within biological samples is critical for assessing gene-editing reagent delivery efficiency, retention, persistence, and distribution within living organisms. Conventional detection methods are effective, yet the expense and lack of scalability for antibody-based affinity reagents limit these techniques for clinical and/or field settings. This necessitates the development of low cost, scalable CRISPR/Cas9 RNP affinity reagents as alternatives or augments to antibodies. Herein, we report the development of the Streptococcus pyogenes anti-CRISPR/Cas9 protein, AcrIIA4, as a novel affinity reagent. An engineered cysteine linker enables covalent immobilization of AcrIIA4 onto glassy carbon electrodes functionalized via aryl diazonium chemistry for detection of CRISPR/Cas9 RNP by electrochemical, fluorescent, and colorimetric methods. Electrochemical measurements achieve a detection of 280 pM RNP in reaction buffer and 8 nM RNP in biologically representative conditions. Our results demonstrate the ability of anti-CRISPR proteins to serve as robust, specific, flexible, and economical recognition elements in biosensing/quantification devices for CRISPR/Cas9 RNP.
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johnston2019usebiosensors Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors Johnston, Robert K;Seamon, Kyle J;Saada, Edwin A;Podlevsky, Joshua D;Branda, Steven S;Timlin, Jerilyn A;Harper, Jason C;
Journal Biosensors & bioelectronics
Year 2019
DOI
S0956-5663(19)30440-3
URL
Keywords
crispr/cas9 electrochemistry affinity reagent anti-crispr protein

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