CRISPR/Cas9-Based Genome Editing Toolbox for Arabidopsis thaliana

CRISPR/Cas9 system has emerged as a powerful genome engineering tool to study gene function and improve plant traits. Genome editing is achieved at a specific genome sequence by Cas9 endonuclease to generate double standard breaks (DSBs) directed by short guide RNAs (sgRNAs). The DSB is repaired by error-prone nonhomologous end joining (NHEJ) or error-free homology-directed repair (HDR) pathways, resulting in gene mutation or sequence replacement, respectively. These cellular DSB repair pathways can be exploited to knock out or replace genes. Also, cytidine or adenine base editors (CBEs or ABEs) fused to catalytically dead Cas9 (dCas9) or nickase Cas9 (nCas9) are used to perform precise base editing without generating DSBs. In this chapter, we describe a detailed procedure to carry out single/multiple gene mutations and precise base editing in the Arabidopsis genome by using CRISPR/Cas9-based system. Specifically, the steps of target gene selection, sgRNA design, vector construction, transformation, and analysis of transgenic lines are described. The protocol is potentially adaptable to perform genome editing in other plant species such as rice.