Advances in guide RNA design for editing plant genomes using CRISPR-Cas systems

This chapter discusses the general rules for selecting target sites for genome editing using the Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology and summarizes the bioinformatics tools that can be used to design sgRNA sequences. The first step in a CRISPR-Cas genome editing experiment is the design of an appropriate sgRNA. To design an sgRNA, some general rules must be followed to maximize on-target editing efficiency and minimize off-target mutation risk. The common sgRNA design is based on generating a multiple sequence alignment, while unique sgRNAs are obtained based on a string comparison algorithm for all possible targets. All sgRNA design tools perform the same basic functions, that is, identifying sgRNAs and off-target sites. To increase the precision of genome editing, more on-target and off-target data are needed to optimize sgRNA design programs. Designing highly specific sgRNAs using bioinformatics programs can help to minimize off-target specificity and increases the likelihood of editing efficiency using CRISPR-Cas and CRISPR-Cpf1 systems.