**3. Discussion**

CRISPR technology, as a powerful and highly e fficient genome editing tool for breeding programs, has been utilized to enable the modification of gene(s) of interest. We report here the CRISPR/Cas9 mutation that potentially could confer a desirable dwarf trait using CRISPR technology. For a long time, plant breeders have used the DELLA gene mutant to reduce plant height [32]. The DELLA protein is one of the main components of the GA signaling pathway and acts as an inhibitor of the GA response. To date, information on dwarfism has shown results in a dominant, semi-dwarf phenotype, such as the observation that the GA signaling repressor DELLA protein or deletion in the N-terminal region suppresses GA signaling [33]. In rice, a total of six slr1-d mutants are known, and these mutants have dark green leaves, reduced internode elongation, and reduced response to GA treatment [28–30,32]. Most slr1-d alleles had 1 bp substitutions, resulting in amino acid substitutions in the conserved TVHYNP motif of *SLR1* (Supplementary Figure S6A). In this study, we generated and characterized six new alleles, namely, slr1-d7~slr1-d12, of the dwarf mutants in rice (Supplementary Figure S6B). These mutants showed the same phenotype for leaf color, GA response, and internode elongation with the previously reported slr1-d1~slr1-d6 mutants [28]. Among these mutants, the slr1-d7 gene had a deletion of three nucleotides, resulting in a serine deletion following core sequencing of the TVHYNP motif. Furthermore, the slr1-d8 mutant showed a 1 bp substitution (+T/+T). These two mutants displayed the most obvious and significant mutant phenotypes. The knockout of the slr1-d8 mutant showed a di fference in plant height compared to the deletion of the serine residue of the slr1-d7 mutant (Figure 1, Supplementary Figure S7). This suggests the importance of the TVHYNP domain

sequence, as the absence of these amino acids a ffects the normal metabolism of the SLR1 protein, but the degree of reduction is weaker than previously reported for the slr1-d6 mutant series [28]. We also performed para ffin sections to investigate cell length and cell layer using slr1-d7, slr1-d8 and WT. As a result, it was found that slr1-d7 and slr1-d8 not only showed significantly reduced cell length, but also node thickening, as cell layers increased as compared to WT (Figure 2). In addition, these dwarf mutants showed a decrease in the whole internode length containing a panicle when compared to WT. This result is similar to the characteristics of *dn*-type rice dwarf mutants reported by Takeda [31] and, as a semi-dominant, a decrease in cell length may be a direct cause of shortening clum length in dwarf mutant plants. In shoot elongation tests of dwarf mutant reaction by exogenous GA3, the length of the secondary leaf sheath was elongated by GA treatment inWT, but not observed in slr1-d7 and slr1-d8 lines. However, there was a di fference between the mutations. These results were similar to those reported in barley *Sln1D* and corn *dwarf 8*, suggesting that a single amino acid deletion or exchange mutation showed an intermediate phenotype depending on plant growth and GAI protein stabilization [13,17,34]. In RNA-seq analysis, the key DEGs related to plant hormone biosynthesis and the signal transduction pathways between slr1-d7 vs. WT and slr1-d8 vs. WT were investigated. DEGs between slr1-d7 vs. WT were down-regulated and included the following: gibberellin-regulated protein 2, gibberellin 2-beta-dioxygenase 8 (*GA2OX8*), E3 ubiquitin-protein ligase (*XERICO*), gibberellin 2-beta-dioxygenase 1 (*GA2OX1*) in GA biosynthesis [35], *ERF03*, *ERF110*, *BBM1* in ethylene biosynthesis [36], and *ILR1* in auxin biosynthesis [37]. In RT-PCR and RNA-seq analysis, the expression levels of two GA-related genes, *GA20OX2* and *GA3OX2*, increased in the edited mutant line compared to WT, suggesting that these genes convert in the GA12 signaling system (Figure 5). The phenomenon of inhibiting cell elongation by altering the GA response due to defects in the signal transduction process of plant hormones was consistent with the results of the *Arabidopsis* mutants [35]. Furthermore, DEGs between slr1-d7 vs. WT were up-regulated and included the following: gibberellin 2-beta dioxygenase 8, *PIF1*, *PIF4*, *GA20OX2*, *GAMYB*, *GA3OX2* in GA biosynthesis [38–43], *ERF109*, *ERF39* in ethylene biosynthesis [36], *LOGL1* in cytokinin biosynthesis [44], *IAA7* in the auxin biosynthesis pathway [45], and *JAR1* in jasmonic acid (JA) biosynthesis [46]. In summary, our results showed that slr1-d7 and slr1-d8 caused a defect in the phytohormone signaling system process and prevented cell elongation. Furthermore, we suggested that the new slr1-d7~slr1-d12 allelic variants are valuable semi-dominant dwarf alleles with potential application value for molecule breeding using the CRISPR/Cas9 system in rice.

#### **4. Materials and Methods**

### *4.1. Plant Materials*

Rice variety Dongjin (*Oryza sativar* L., ssp. Japonica) was used for transformation experiments. Plants were grown in GMO greenhouse facilities and rice fields at Hankyong National University in Korea. Harvested seeds were dried to ~14% moisture content and kept in dry conditions at 4 ◦C.

#### *4.2. CRISPR*/*Cas9 Vector Construction and Rice Transformation*

SgRNAs were designed as described in Park et al. [47] to target the TVHYNP motif. The TVHYNPSD amino acid sequence of the *SLR1* gene is encoded by ACCGTGCACTACAACCCCTCGGAC, and the target sequence ACCCCTCGGACCTCTCCCTCCTGG with TGG as the PAM was selected. The 20nt sgRNA sca ffold sequence was synthesized by Bioneer co., LTD (Dajeon, Korea). The slr-sgRNA templates were annealed using two primers, 5--ggcagACCCCTCGGACCTCTCCTCC-3- and 5--aaacGGAGGAGAGGTCCGAGGGGTc-3-, and cloned into an *Aar*I-digested *OsU3*:pBOsC binary vector. The Ti-plasmid vector for sgRNA expression, OsU3:*slr1*-sgRNA/pBOsC, and its flanking sequences were confirmed by the Sanger sequencing method and mobilized into *Agrobacterium-tumefaciens* strain EHA105. Transgenic plants were regenerated following a previously described protocol [48]. To confirm the transgene, the independent and transformed lines were

analyzed by PCR. Plants derived from tissue culture were rooted and potted into 7 cm pots placed in the glasshouse and gradually acclimatized to the glasshouse conditions.

#### *4.3. Targeted Deep Sequencing and Mutation Analysis*

Total DNA extraction from plant tissues was performed using the DNA Quick Plant Kit (Inclone, Korea). Targeted deep sequencing analysis was performed following the method described by Jung et al. [46]. All primers used for targeted deep sequencing are listed in Supplementary Table S1. Paired-end read sequencing by PCR amplicons was produced with MiniSeq (Illumina, San Diego, CA, USA). All data derived from MiniSeq were analyzed by Cas-Analyzer (http://www.rgenome.net/casanalyzer), as previously reported by Park et al. [49].

#### *4.4. RNA-Seq and Data Analysis*

To investigate the transcriptome of edited lines obtained by the *OsSLR1* gene via the CRISPR/Cas9 system, WT, slr1-d7(T/T), and slr1-d8 (−3/−3) plants were used for RNA-seq analysis. Four-week-old leaf tissues were used for RNA extraction, as previously reported by Wang et al. [50]. RNA concentration (A260/A280 and A260/A230) was measured with spectrophotometry (Nanodrop 2000, Thermo Scientific, Hudson, NH, USA). A Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to evaluate the RNA qualities. Leaf samples of 100 mg were collected from three plants (WT, slr1-d7, and slr1-d8) for RNA-seq analysis. RNA-sequencing was carried out by Macrogen (Seoul, Korea, https: //dna.macrogen.com/). Clean reads were produced by removing low-quality reads and mapped to the reference genome (https://plants.ensembl.org/) using TopHat2 (https://ccb.jhu.edu/software/tophat) [51]. Based on location information of the mapped reads, gene expression levels were normalized to reads per kilobase per million mapped reads (RPKM). DEG analyses between the edited plant RNA (slr1-d7, slr1-d8 and WT) were performed using the standard fold change (FC) ≥2 and FDR <0.05. GO analysis was performed as previously reported by Chow et al. [52].

#### *4.5. Validation Test of Selected DEGs*

To validate the accuracy of the RNA-sequencing data, qRT-PCR was conducted on twenty-one selected genes. The slr1-d7 and slr1-d8 lines were assessed according to WT, and relative gene expression levels were normalized by the *Actin* gene (XM\_015761709). All assays for each gene were performed in triplicate with the same conditions and the RNA-seq data were deposited into the NCBI database.

### *4.6. GA3 Treatment*

The slr1-d7, slr1-d8, and WT seedlings grown in pots for 4 weeks were sprayed with 50 μM GA3 (Sigma-Aldrich, Seoul, Korea). The stock solution of GA3 was dissolved in ethanol and added to autoclaved water after cooling to approximately 60 ◦C to make the final 50 μM solution. The WT plants were treated with water containing equal amounts of ethanol.

### *4.7. Light Microscopy*

For the para ffin section, stems and leaves were harvested from slr1-d7, slr1-d8, and WT plants. First, stem tissues were treated with 15% hydrofluoric acid, followed by dehydration with 70% ethanol, removal of it, infiltration, and embedding. For imaging, a 10 μm microtome section was placed on glass slides and floated in a 37 ◦C water bath containing deionized water. The sections were floated onto clean glass slides and microwaved at 65 ◦C for 15 min. Following this, the tissues were bound to the glass and each slide was used in chemical staining immediately.

**Supplementary Materials:** Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/15/ 5492/s1. **Figure S1.** Nucleotide and amino acid sequences of the *SLR1* gene in rice. **Figure S2.** Amino acid sequence alignment of the coding region of the *SLR1* gene from *Oryza sativa*, *Sorghum bicolor, Zea mays, Panicum miliaceum* and *Triticum aestivum*. **Figure S3.** Confirmation of the e fficiency of sgRNA using T7-endonuclease I

enzyme. **Figure S4.** CRISPR/Cas9 binary vector construction and rice transformation. **Figure S5.** Confirmation of the mutant phenotype and the conserved domain region of the DELLA protein. **Figure S6.** CRISPR/Cas9-induced mutations in the *OsSLR1* gene and the phenotype of the edited plants. **Figure S7.** Appearance of the panicle, leaves and grains after harvesting slr1-d7 and slr1-d8 lines compared to WT. **Table S1.** Design of sgRNAs for CRISPR genome editing on the *OsSRL1* gene in rice. **Table S2.** Mutation rate and edited plant types for the *OsSLR1* gene using the CRISPR/Cas9 system. **Table S3.** GO enrichment analysis of DEGs by RNA-seq analysis. **Table S4.** Key DEGs of up- and down-regulated genes related to phytohormone biosynthesis and signaling transduction pathway by RNA seqs in WT vs slr1-d7 and WT vs slr1-d8 lines. **Table S5.** List of primers and gene sequences in DEGs, correlation analysis of gene expression pattern by RNA-Seq and qRT-PCR.

**Author Contributions:** Formal analysis, Y.J.J., J.H.K., H.J.L., D.H.K. and J.Y.; investigation, Y.-G.C.; resources, S.B. and K.K.K.; software, H.J.L., J.Y. and S.B.; supervision, K.K.K.; writing-original draft, Y.J.J.; writing-review and editing, K.K.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by "Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01477203)" Rural Development Administration, Korea.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
