General guidelines for CRISPR/Cas-based genome editing in plants.

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) technology is a versatile genome editing tool that has been used to improve agriculturally important plant traits. Due to its precision, CRISPR/Cas9 is more effective than either conventional plant breeding methods or standard genetic engineering approaches for the rapid development of new varieties resilient to climate change. In addition to knowledge in tissue culture-based plant transformation, effective gene-specific single guide RNA (sgRNA) design, prediction of its off-target effect and utilization of vectors, promoters, Cas proteins and terminators is required for CRISPR/Cas9. Various bioinformatics tools are available for the best sgRNA design and screening of the off-targets. Various tools are used in the delivery of CRISPR/Cas components into cells and the genome. Moreover, some recent studies proved the simultaneous silencing of different paralogs in the same family or several genes working in the same pathway by using multiple-target sgRNA designs. This review summarizes the type of promoters, Cas proteins, recognition sequences, and terminators available for the development of knock-out and overexpression plant lines. It also provides a general guideline for the development of genome-edited plants from the design of sgRNAs to the selection of non-transgenic genome-edited T2 generation.