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

Journal Article · Sun Sep 15 00:00:00 EDT 2019 · Biosensors and Bioelectronics

DOI:https://doi.org/10.1016/j.bios.2019.111361· OSTI ID:1559508

Johnston, Robert K. ^[1]; Seamon, Kyle J. ^[2]; Saada, Edwin A. ^[2]; Podlevsky, Joshua D. ^[2]; Branda, Steven S. ^[2]; Timlin, Jerilyn A. ^[2]; Harper, Jason C. ^[2]

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1. Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
2. Sandia National Lab. (SNL-CA), Livermore, CA (United States)

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.

• Research Organization: Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
• Sponsoring Organization: USDOE National Nuclear Security Administration (NNSA)
• Grant/Contract Number: AC04-94AL85000
• OSTI ID: 1559508
• Alternate ID(s): OSTI ID: 1778458
• Report Number(s): SAND2019-9186J; 678234
• Journal Information: Biosensors and Bioelectronics, Vol. 141, Issue C; ISSN 0956-5663
• Publisher: Elsevier
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES