A Comprehensive Protocol for Assembly of Multiple gRNAs into a Direct Vector for Genome Editing in Tomato

Valluri V Satyavathi; Kunnappady Princy; Neha Gupta; Narasimha Rao Nizampatnam; Rameshwar Sharma; Yellamaraju Sreelakshmi

doi:10.1007/978-1-0716-3782-1_19

A Comprehensive Protocol for Assembly of Multiple gRNAs into a Direct Vector for Genome Editing in Tomato

Methods Mol Biol. 2024:2788:317-335. doi: 10.1007/978-1-0716-3782-1_19.

Authors

Valluri V Satyavathi¹, Kunnappady Princy¹, Neha Gupta¹, Narasimha Rao Nizampatnam², Rameshwar Sharma¹, Yellamaraju Sreelakshmi¹

Affiliations

¹ Repository of Tomato Genomics Resources, Department of Plant Sciences, University of Hyderabad, Hyderabad, India.
² ATGC Biotech Pvt. Ltd, Hyderabad, India.

PMID: 38656523
DOI: 10.1007/978-1-0716-3782-1_19

Abstract

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas 9 (CRISPR-associated protein 9) is a robust DNA-encoded, RNA-mediated sequence-specific nuclease system widely used for genome editing of various plants. Although there are many reports on the assembly of gRNAs and plant transformation, there is no single resource for the complete gene editing methodology in tomato. This chapter provides a comprehensive protocol for designing gRNAs, their assembly into the vector, plant transformation, and final mutant analysis in tomato.

Keywords: CRISPR Cas9; Multiple gRNA; Plant tissue culture; Plant transformation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems*
Gene Editing* / methods
Genetic Vectors* / genetics
Genome, Plant
Plants, Genetically Modified / genetics
RNA, Guide, CRISPR-Cas Systems* / genetics
Solanum lycopersicum* / genetics
Transformation, Genetic

Substances

RNA, Guide, CRISPR-Cas Systems