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

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 recognitionmore » elements in biosensing/quantification devices for CRISPR/Cas9 RNP.« less

Authors:

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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+ Show Author Affiliations

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

Publication Date:

Sun Sep 15 00:00:00 EDT 2019

Research Org.:

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

Sponsoring Org.:

USDOE National Nuclear Security Administration (NNSA)

OSTI Identifier:

1559508

Alternate Identifier(s):

OSTI ID: 1778458

Report Number(s):

SAND2019-9186J
Journal ID: ISSN 0956-5663; 678234

Grant/Contract Number:  

AC04-94AL85000

Resource Type:

Accepted Manuscript

Journal Name:

Biosensors and Bioelectronics

Additional Journal Information:

Journal Volume: 141; Journal Issue: C; Journal ID: ISSN 0956-5663

Publisher:

Elsevier

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES