Accession-Dependent CBF Gene Deletion by CRISPR/Cas System in Arabidopsis

Abstract

The CRISPR/Cas system became a powerful genome editing tool for basic plant research and crop improvement. Thus far, CRISPR/Cas has been applied to many plants, including Arabidopsis, rice and other crop plants. It has been reported that CRISPR/Cas efficiency is generally high in many plants. In this study, we compared the genome editing efficiency of CRISPR/Cas in three different Arabidopsis accessions [Col-0, Ler, and C24RDLUC (C24 accession harboring the stress-responsive RD29A promoter-driven luciferase reporter)]. For the comparison, we chose to target the cold-responsive <i>C-repeat/DRE-Binding Factor</i> (<i>CBF</i>) genes. <i>CBF1</i>, <i>CBF2</i>, and <i>CBF3</i> genes are tandemly located on Arabidopsis chromosome 4 with redundant functions as the key transcription factors functioning in cold stress signaling and tolerance. Due to the close proximity of these <i>CBF</i>s on the chromosome, it is impossible to generate <i>cbf1, cbf2, cbf3</i> triple mutants (<i>cbf123</i>) by traditional genetic crosses. Therefore, using the CRISPR/Cas tool, we aimed to generate <i>cbf123</i> mutants and compared the genome editing efficiency in different Arabidopsis accessions. Among the accessions, Ler was the most resilient to the CRISPR/Cas deletion with the lowest gene deletion ratio in both T1 and T2 generations. Interestingly, while C24RDLUC showed a high <i>CBF123</i> deletion frequency in T2 only when the gene deletion was observed in T1 generation, Col-0 displayed high ratios of the <i>CBF123</i> deletions in T2 regardless of the presence or absence of the <i>CBF123</i> deletion in T1. Isolated <i>cbf123</i> mutants in C24RDLUC background showed no expression of <i>CBF1</i>, <i>CBF2</i>, and <i>CBF3</i> genes and proteins with reduction in the <i>CBF</i> target gene expression under cold stress.