1. Use of anti-CRISPR protein AcrIIA4 as a capture ligand for CRISPR/Cas9 detection.
- Author
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Johnston RK, Seamon KJ, Saada EA, Podlevsky JD, Branda SS, Timlin JA, and Harper JC
- Subjects
- CRISPR-Cas Systems, Immobilized Proteins chemistry, Ligands, Models, Molecular, Bacterial Proteins analysis, Bacteriophages chemistry, Biosensing Techniques methods, CRISPR-Associated Protein 9 analysis, Streptococcus pyogenes chemistry, Viral Proteins chemistry
- 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., (Copyright © 2019 Elsevier B.V. All rights reserved.)
- Published
- 2019
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