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 C-repeat/DRE-Binding Factor (CBF) genes. CBF1, CBF2, and CBF3 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 CBFs on the chromosome, it is impossible to generate cbf1, cbf2, cbf3 triple mutants (cbf123) by traditional genetic crosses. Therefore, using the CRISPR/Cas tool, we aimed to generate cbf123 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 CBF123 deletion frequency in T2 only when the gene deletion was observed in T1 generation, Col-0 displayed high ratios of the CBF123 deletions in T2 regardless of the presence or absence of the CBF123 deletion in T1. Isolated cbf123 mutants in C24RDLUC background showed no expression of CBF1, CBF2, and CBF3 genes and proteins with reduction in the CBF target gene expression under cold stress.

Epigenetic switch from repressive to permissive chromatin in response to cold stress

“…After 2 min of centrifugation at 16,000 × g , the supernatant was transferred to a siliconized tube, and 10 μL of the appropriate antibody was added. Antibodies used were anti-AcH3 (Millipore), antiacetylated H3K9/K14 (Upstate Biotechnology), anti-HOS15 ( 66 ), anti-CBFs ( 70 ), and anti-HD2C (Agrisera). After incubation overnight with rotation at 4 °C, 60 μL SS DNA/Protein A agarose was added and incubation continued for 2 h. The agarose beads were then washed with 1 mL of each of the following: two times lysis buffer, one time LNDET buffer [0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris (pH 8)], and three times TE buffer [10 mM Tris⋅HCl (pH 8), 1 mM EDTA].…”

Ares-GT: Design of guide RNAs targeting multiple genes for CRISPR-Cas experiments

“…The first three members of this family are closely located in less than 8 Kb in chromosome 4 (Fig 1A), making extremely difficult to obtain a triple mutant by classical crossing strategy. This has been recently achieved by CRISPR/Cas9-induced mutagenesis [23] using two sgRNAs that the authors selected by manual evaluation of sequence alignments, manual selection of candidates, and specificity verification with CRISPR-P [1]. I used the A. thaliana genomic coding sequences (TAIR v10) of the four CBF genes as a multiple query in ARES-GT, to search for candidate sgRNAs using both Cas9 and Cas12a.…”

Ares-GT: design of guide RNAs targeting multiple genes for CRISPR-Cas experiments

“…The first three members of this family are closely located in less than 8 Kb in chromosome 4 ( Figure 1A), making extremely difficult to obtain a triple mutant by classical crossing strategy. This has been recently achieved by CRISPR/Cas9-induced mutagenesis (Cho et al, 2017) using two sgRNAs that the authors selected by manual evaluation of sequence alignments, manual selection of candidates, and specificity verification with CRISPR-P (Lei et al, 2014). I used the A. thaliana genomic coding sequences (TAIR v10) of the four CBF genes as a multiple query in ARES-GT, to search for candidate sgRNAs using both Cas9 and Cas12a.…”

“…In total, 10 Cas9 and 5 Cas12a candidates were identified that match more than one CBF gene and did not present any offtarget outside CBF genes ( Figure 1B, 1C). Among them were included the two sequences previously reported (Cho et al, 2017), corresponding to Cas9CBF1_015 and Cas9CBF2_124 in this work.…”

“…Location of Cas9 (B) and Cas12a (C) candidates with multiple CBF gene targets. (*) Asterik marks candidates corresponding with previously reported sgRNAs(Cho et al, 2017).…”