The sonication-assisted whisker method enables CRISPR-Cas9 ribonucleoprotein delivery to induce genome editing in rice

Abstract

CRISPR/Cas9-based genome editing represents an unprecedented potential for plant breeding. Unlike animal cells, plant cells contain a rigid cell wall, genome editing tool delivery into plant cells is thus challenging. In particular, the delivery of the Cas9-gRNA ribonucleoprotein (RNP) into plant cells is desired since the transgene insertion into the genome should be avoided for industrial applications in plants. In this study, we present a novel RNP delivery approach in rice. We applied the sonication-assisted whisker method, conventionally developed for DNA delivery in plants, for RNP delivery in rice. Combined with marker gene delivery, we successfully isolated OsLCYβ genome-edited lines generated by RNPs. The calli and regenerated shoot of the OsLCYβ mutant showed abnormal carotenoid accumulation. In addition, we also detected, although at a low frequency, genome editing events in rice calli cells by RNP delivery using the sonication-assisted whisker method without any additional. Therefore, the sonication-assisted whisker method could be an attractive way to create RNP-based genome-edited lines in plants.

Figure 1

Overview of the experimental procedure of CRISPR/Cas9 RNP delivery with the sonication-assisted whisker method using selection marker-harboring plasmids. The recombinant SpCas9-NLS and in vitro transcribed gRNA were mixed to prepare the RNPs. The RNPs were mixed with the whiskers, the selection marker-harboring, plasmids, and specific amounts of rice embryonic suspension cells (250 packed cell volume [PCV]). The calli mixed with whiskers and RNPs were ultrasonicated by a sonicator. Sonication-treated cells were washed with the R2 medium and incubated without antibiotics for the recovery culture. After the 6-day recovery culture, we conducted a hygromycin-based selection of the transformed calli for 1–2 weeks. Selected calli were split into fragments, one of which was subjected to DNA extraction and genome sequence analysis using Amplicon sequencing (Amplicon-seq). The rest of the selected calli, not used for genome DNA analyses, were incubated further and transferred into a regeneration medium to obtain the shoot. OsPDS exon12 was selected as the genome editing target (inlet). The target sequence of the gRNA and the PAM sequence were marked as red and blue, respectively. The restriction enzyme PstI recognition site is underlined. The in vitro transcribed gRNA sequence is also indicated in the inlet.

Figure 2

Representative results of OsPDS locus genome editing in rice by RNP delivery using the sonication-assisted whisker method. (a) A representative image of white-colored calli after the incubation of the selection medium for 2 weeks; (b) genome sequences of the OsPDS with targeted mutations in the whisker-treated calli after the incubation of the selection medium for 2 weeks. DNA from independently selected calli were analyzed by Amplicon-seq. The gRNA target sequence and the PAM sequence are colored in red and blue, respectively. The insertion bases are highlighted in magenta. The numbering on the left corresponds to independent calli (e.g., #6 has two types of mutations in one callus). The genotypes are also summarized in Supplementary Table 1. The percentages in the right of the sequences indicate the percentage of the reads with mutation/the reads with no mutations; (c) a representative image of regenerated plants with green- (control, left), and white-colored shoots (genome-edited, right).

Figure 3

Isolation of OsLCYβ and OsLCYε mutants using RNP delivery by the sonication-assisted whisker method. (a) gRNA design for OsLCYβ genome editing. OsLCYβ exon1 was selected as the genome editing target. The target sequences of each gRNA and the PAM sequence are marked in red and blue, respectively. The in vitro transcribed gRNA sequence is also indicated; (b) gRNA design for OsLCYε genome editing. OsLCYε exon1 was selected as the genome editing target. The coloration of the schematic image is the same as those of (a). (c) Detailed mutations detected in the selected calli of the OsLCYβ or OsLCY ε mutants. The name of gRNAs is indicated in each sequence alignment. The genome sequence with no mutation is described as “WT.” The insertions and the PAM sequence are highlighted in magenta and blue, respectively. The gRNA target sequence is colored with red. Dashed lines indicate the putative DSB sites.

Figure 4

Phenotypic analysis of the OsLCYβ mutant generated by RNP delivery using the sonication-assisted whisker method. (a) Representative images of non-RNP-treated calli (left) and genome-edited lines of LCYβ-2 #2 after the 2-week selection (right); (b) the callus of LCYβ-2 #2 showed clear red pigmentation; (c) representative images of the shoot from the non-RNP-treated calli (left) and that from the genome-edited calli of LCYβ-2 #2; (d) quantification of the carotenoids in the LCYβ-2 #2 calli, **indicate statistical significance analyzed by the Welch Two Sample t-test between WT and LCYβ-2 #2 (p < 0.05).