“CRISPR Cas12a and 13a based Plant Virus Detection”

   Plant viruses are responsible for many commercially important plant diseases, infecting a wide range of plant species and resulting in severe quality and yield losses in diverse crops. Plant diseases are estimated to cause 10 to 15% reductions in global crop yields annually, with 47% of these losses caused by viruses. Viral infections in plants threaten food security. Thus, simple and effective methods for virus detection are required to adopt early measures that can prevent virus spread.

Current viral diagnostics typically include a procedure to amplify a viral genome fragment, such as the polymerase chain reaction (PCR) and its derivative approaches or to detect viral proteins, such as the enzyme-linked immunosorbent assay. These methods require sophisticated equipment, infrastructure, and skilled technical staff. Furthermore, isothermal amplification-based methods such as loop-mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA) have been applied to detect several plant viruses. To solve the specificity issue, systems based on CRISPR and Cas proteins have been repurposed in recent years for diagnostic applications in combination with isothermal amplification methods. To address these challenges, the variants of CRISPR-Cas viz., Cas12a, and Cas13a were explored. Being different from Cas9, Cas12a and Cas13a intriguingly exhibit collateral, nonspecific activities on random ssDNA or ssRNA respectively upon target recognition. Utilizing the collateral activity, Cas13a and Cas12a are respectively explored for nucleic acid detection1.

The CRISPR-Cas12a and Cas13a systems to detect three RNA viruses, namely, Tobacco mosaic virus, Tobacco etch virus, and Potato virus X, in Nicotiana benthamiana plants. The CRISPR-Cas12a system to detect viral DNA amplicons generated by PCR or isothermal amplification, and finally, the CRISPR-Cas13a/d system to directly detect viral RNA, thereby avoiding the necessity of a preamplification step and obtaining a readout that scales with the viral load2. CRISPR based nucleic acid diagnostic method utilizing the CRISPR–Cas12a system for detecting two geminiviruses, tomato yellow leaf curl virus (TYLCV) and tomato leaf curl New Delhi virus (ToLCNDV)3.

CRISPR- Cas12a and Cas13a are revolutionizing plant virus detection, offering a powerful and versatile toolbox for protecting our crops and ensuring food security. These tools are being explored for field-based monitoring of agricultural crops, aiding in rapid disease control and preventing yield losses. With continued advancements, these innovative tools hold immense potential for the future of plant health management.

References:

 1YIN, L., MAN, S., YE, S., LIU, G. AND MA, L., 2021, CRISPR-Cas based virus detection: Recent advances and perspectives. j. biosens. bioelectron., 193, 113541.

 2MARQUES, M.C., SANCHEZ-VICENTE, J., RUIZ, R., MONTAGUD-MARTINEZ, R., MARQUEZ-COSTA, R., GOMEZ, G., CARBONELL, A., DAROS, J.A. AND RODRIGO, G., 2022, Diagnostics of infections produced by the plant viruses TMV, TEV, and PVX with CRISPR-Cas12 and CRISPR-Cas13. ACS Synth. Biol., 11(7), 2384-2393. 

3MAHAS, A., HASSAN, N., AMAN, R., MARSIC, T., WANG, Q., ALI, Z. AND MAHFOUZ, M.M., 2021, LAMP-coupled CRISPR–Cas12a module for rapid and sensitive detection of plant DNA viruses. Viruses, 13(3), 466.