TL;DR
This protocol covers both single-gRNA (sgRNA) and dual-gRNA directed knockout systems and describes the procedure for constructing an Auxin-Inducible Degron (AID) knock-in-mediated system to induce the rapid degradation of a target protein in cells, thereby investigating its function.
This chapter outlines a comprehensive methodology for the design, assembly, and functional assessment of CRISPR/dCas9 systems optimized for tomato to investigate pathogen-associated responses.
The application of genome editing, CRISPR/Cas9 has revolutionized plant breeding by enabling precise, efficient, and targeted modification of native genes, significantly accelerating the development of improved agronomic traits of crops. Therefore, CRISPR/Cas9 technology currently the most extensively used genome editing technique worldwide because of its simple design, cost-effectiveness, high efficiency, good reproducibility, high engineering feasibility, ability to create gene knockout, RNA editing, and quick cycle. It is used to knock in or knock out genes of interest and for generating models for genetic studies. The main components of the CRISPR/Cas9 system are an RNA-guided Cas9 endonuclease and a single-guide RNA (sgRNA). The workflow of CRISPR/Cas9 gene editing comprises selecting target sites, designing and synthesizing sgRNA, introducing transformation constructs or ribonucleoprotein (RNP) in plant cells, followed by transformation and identification of edited lines. This approach bypasses the formal regulations on GMOs, thus encouraging the widespread adoption RNA-guided gene editing in agricultural sciences and biotechnology. The system is now being utilized in the biofortification of cereal crops such as rice, wheat, barley, and maize, including vegetable crops such as potato and tomato. The world's first genome-edited rice varieties are DRR Dhan 100 (Kamala) and Pusa DST Rice 1 developed by the Indian Council of Agricultural Research (ICAR), New Delhi, India in 2025 with the objective of bringing about revolutionary changes in terms of higher production, climate adaptability, and water conservation. The CRISPR/Cas9-based crop genome editing has been utilized in imparting/producing qualitative enhancement in aroma, shelf life, sweetness, and quantitative improvement in starch, protein, gamma-aminobutyric acid (GABA), oleic acid, anthocyanin, phytic acid, gluten, and steroidal glycoalkaloid contents. Some varieties have even been modified to become disease and stress-resistant. Therefore, CRISPR/Cas9 is aiding in developing climate-ready crops and improving crop quality parameters such as appearance, palatability, nutritional components, and other preferred traits. Gene editing tools are used to generate changes to the native genetic material. Unlike GMOs, which introduce novel configurations of genetic materials typically derived from other organisms, gene editing methods modify existing genetic material in ways that can yield beneficial outcomes.
Natural variation in PAM recognition among SaCas9 orthologs is analyzed and StaCas9 is identified as a compact and efficient nuclease recognizing an NNG PAM, establishing StaCas9 as a high-performance genome-editing tool for therapeutic applications.
The CRISPR/Cas9 system is utilized to generate a homozygous Mt4 knockout (Mt4-/-) mouse embryonic stem cell (mESC) line that maintains normal morphology, pluripotency, and the ability to differentiate into all three germ layers.
Traditional methods of diagnosis of TB and mechanisms and challenges related to CRISPR-Cas are included, including the novel technology CRISPR-Cas system for diagnosis, treatment, and the development of new TB vaccines.
This review summarizes CRISPR principles, highlights technical breakthroughs and crop applications that mitigate biotic and abiotic stresses, and outlines practical challenges and future directions necessary for responsible deployment in agriculture.