Monitoring single-cell bioluminescence of <i>Arabidopsis</i> leaves to quantitatively evaluate the efficiency of a transiently introduced CRISPR/Cas9 system targeting the circadian clock gene <i>ELF3</i>

Abstract

The rapid assessment of gene function is crucial in biological research. The CRISPR/Cas9 system is widely used as a tool for targeted gene editing in many organisms including plants. Previously, we established a transient gene expression system for investigating cellular circadian rhythms in duckweed. In this system, circadian reporters and clock gene effectors-such as overexpressors, RNA interference (RNAi), and CRISPR/Cas9-were introduced into duckweed cells using a particle bombardment method. In the present study, we applied the CRISPR/Cas9 system at a single cell level to Arabidopsis thaliana, a model organism in plant biology. To evaluate the mutation induction efficiency of the system, we monitored single-cell bioluminescence after application of the CRISPR/Cas9 system targeting the ELF3 gene, which is essential for robust circadian rhythmicity. We evaluated the mutation induction efficiency by determining the proportion of cells with impaired circadian rhythms. Three single guide RNAs (sgRNAs) were designed, and the proportion of arrhythmic cells following their use ranged from 32 to 91%. A comparison of the mutation induction efficiencies of diploid and tetraploid Arabidopsis suggested that endoreduplication had a slight effect on efficiency. Taken together, our results demonstrate that the transiently introduced CRISPR/Cas9 system is useful for rapidly assessing the physiological function of target genes in Arabidopsis cells.

Adaptive diversification in the cellular circadian behavior of Arabidopsis leaf- and root-derived cells

“…The mean periods of leaf-derived cells (Supplementary Table. 1) were comparable to those of detached leaves under DD conditions 18,22,27 , whereas those of root-derived cells were shorter than those reported for roots under DD conditions 11,21,24,35 . Interestingly, period lengths are highly variable even for same roots, and the involvement of input signals from neighboring cells in the determination of period has been proposed 24 .…”

“…In contrast, leaf-derived cells sustained the amplitudes of the rhythms (Fig. 1), and the sustained amplitudes were also observed in detached leaves under DD conditions 18,22 . In plants, low temperature disrupts circadian rhythmicity 36,37 , and we observed highly unstable circadian rhythms of protoplast-derived cells at 12°C (Fig.…”

Low-temperature and circadian signals are integrated by the sigma factor SIG5

“…3a and Supplementary Data 1 and 2). We transiently expressed SIG5::LUCIFERASE in the wild type and hy5, hyh and hyh5 hyh mutants using particle bombardment 35,36 and found that SIG5 promoter activity was reduced substantially in the hy5 and hy5 hyh double mutants, and partially in the hyh mutant, relative to the wild type (Extended Data Fig. 3c).…”

“…A SIG5::LUCIFERASE reporter construct (pGREENII0229 SIG5:: LUCIFERASE 24 ) was expressed transiently in Arabidopsis seedlings using methods similar to those used previously 35,36 . Briefly, 5 µg of pGREENII0229 SIG5::LUCIFERASE or pB7WG2.0-GFP (green fluorescent protein (GFP) positive control for transformation 100 ) was combined with 25 µl of 1 nm gold particle suspension (Bio-Rad), 25 µl of 2.5 M CaCl 2 and 10 µl of 0.1 M spermidine (Sigma), and incubated on ice for 30 min.…”