**2. Results**

#### *2.1. Editing of the TVHYNP Domain Encoding the OsSLR1 Gene and the CRISPR*/*Cas9 System*

According to the structure of the *OsSLR1* gene, DELLA and TVHYNP domains are well conserved at the N-terminus (Figure 1A, Supplementary Figure S1 and S2). Sixteen mutants were identified by single guide RNA (sgRNA) region which targeted the *OsSLR1* gene in the positive transgenic T0 plants (Supplementary Table S1, Figure S3 and S4). Deep sequence analyses detected 6 homozygous mutations, 2 heterozygous mutations, and 8 bi-allelic mutations (Supplementary Table S2). All the T0 mutants were dwarf, producing many tillers. Six homozygous mutants were identified, among which four were characterized by few bp deletions and two by a few bp insertions. Specifically, the following were observed: a 3-bp deletion and mutant named slr1-d7, a 1-bp deletion that was designated as slr1-d8, a 5-bp deletion named slr1d9 and a 14-bp deletion called slr1-d10. The insertion mutants were a T-insertion named slr1d11 and a C-insertion named slr1-d12 (Figure 1B). Theoretically, the slr-d7 mutant encodes a protein without serine (Ser, S) and the slr1-d8~slr1-d12 are knockout mutants with a stop codon that cannot encode the protein (Figure 1C). A single-base deletion and insertion are predicted to cause a frameshift, resulting in the knockout of the *OsSLR1* gene. However, all the

mutations did not affect the core sequence TVHYNP domain of the *OsSLR1* gene. The sgRNA was also investigated using Cas-OFFinder (http://www.rgenome.net/cas-offinder/) [27], and two potential off-target sites were chosen. Interestingly, no mutations were detected in these loci. These mutations are either untranslated or modified SLR1 proteins and have different mutant sites compared to the previously reported slr1-d1~slr1-d6 allele [28–30].

**Figure 1.** Genome editing in the rice *OsSLR1* gene. (**A**) Design of single guide RNA (sgRNA) sites in the TVHYNP motif; the nuclease cleavage site is represented by the red arrow and the Protospacer Adjacent Motif (PAM) (NGG) appears in blue. DELLA protein organization representing the conserved domains. (**B**) Nucleotide sequence alignment by deep sequence analysis of the sgRNA target region in six mutant lines of transformed rice plants. Deletion and insertion indicated by dash and red letters, respectively. (**C**) Amino acid sequences of the target region in six mutant lines.

#### *2.2. New Allelic Slr-d7~Slr-d8 Mutant Plants Showed Dwarfing*

The new dwarf mutants (slr1-d7~slr1-d12) showed several deficiencies in addition to reducing plant size (Supplementary Figure S5). Compared to the wild type (WT), these mutants had a slow growth rate, showed dwarfed and shriveled leaves (Figure 2A). The stomata are the key channels that regulate gas exchange and water evaporation in the leaves. As a result of observing stomata sizes by SEM (scanning electron microscopy) images, the slr1-d7 and slr1-d8 lines were smaller than that of WT (Figure 2B). To observe cytological differences in the stem internodes of these mutants, paraffin sections of the stem internodes were investigated from two mutants (slr1-d7 and slr1-d8) and WT. These dwarf mutants showed that the cell size was significantly reduced, and the internode thickened as the cell layer was increased (Figure 2C). In addition, the length of all internodes of the slr1-d7 and slr1-d8 lines were reduced compared to WT (Figure 2D). These results are similar to the characteristics of *dn*-type rice dwarf mutants previously reported by Takeda [31]. Thus, slr1-d7 and slr1-d8 lines were semi-dominant dwarf mutants, indicating that a decrease in cell length may be a direct cause of shortened culm length in dwarf mutant plants. Furthermore, to know the cause of dwarfism, the length of the leaf sheath was measured according to GA3 concentration treatment in slr-d7, slr-d8, and WT. The results showed that the slr-d7 and slr-d8 variants produced a more extended leaf sheath following GA3 treatment, but a reduced length extension compared to WT (Figure 2E).

**Figure 2.** Phenotypic analysis of wild-type (WT) and slr1 mutant plants. (**A**) Phenotype of mature WT and mutant plant lines. (**B**) SEM (scanning electron microscopy) images of rice stomata in slr1-d7, slr1-d8 and WT. (**C**) Longitudinal tissue sections of the main stem at the mature stage in WT and slr1 mutant using paraffin section. Bar: 100 μm. (**D**) Length of internodes in slr1-d7, slr1-d8 and WT. (**E**) Elongation of the second leaf sheath of slr1-d7 and slr1-d8 in response to exogenous treatment with different concentrations of GA3. Error bars are SD from the mean (*n* = 3).

#### *2.3. Altered Transcriptome Profiling in Slr-d7 and Slr-8 Mutants*

To understand the impact of dwarfism on gene expression at the whole-genome level, RNA-Seq was conducted to detect transcription profiling changes in WT, slr1-d7 and slr1-d8 lines. RNA-seq results showed that gene expression was altered significantly between WT and the slr1-d7 and slr1-d8 lines (Figure 3A). There are 214 genes upregulated and 154 genes downregulated in the slr1-d7 mutant compared with WT plants. By comparison, 334 genes were upregulated and 104 genes were downregulated in the slr1-d8 line (Figure 3B). Venn diagram analysis revealed 806 genes expressed in both WT and slr1-d7 or slr1-d8 mutants, which may explain the effects of knocking out *SLR1* on plants (Figure 3B,C). Gene ontology (GO) enrichment analysis of the 806 annotated up- and down-regulated genes identified 193 significantly (false discovery rate (FDR < 0.05)) enriched GO terms for the biological process, cellular component, and molecular function categories (Figure 4). Within the biological process category, the enriched differentially expressed genes (DEGs) were mainly associated with the response to the oligopeptide transport (GO:0006857), the intracellular protein transport (GO:0006886), karrikin (GO:0080167), and salt stress (GO:0009651). Within the cellular component category, the enriched DEGs were mainly associated with the plasma membrane (GO:0005886), the membrane (GO:0016020), cytosol (GO:0005829), and the integral component of the membrane (GO:0016021). Within the molecular function category, the DEGs were associated with protein serine/threonine kinase activity (GO:0004674), ATP binding (GO:0005524), and protein binding (GO:0005515) (Supplementary Table S3). To confirm the results from the RNA-seq analysis, 38 DEGs in the enriched GO terms were selected in the slr1-d7 and slr1-d8 lines, and their expression levels were confirmed by qRT-PCR analysis. The qRT-PCR results showed that the transcription levels of these genes were consistent with the RNA-seq results (Figure 5). These results indicated that dwarfism of the slr1-d7 and slr1-d8 lines mediates gene expression levels involved in regulating the plant hormone (GA, salicylic acid (SA), jasmonate (JA), IAA, cytokinin (CT) and ethylene (ET)) metabolism, signal transduction and transport (Supplementary Table S4).

**Figure 3.** Global gene expression changes in knockout *OsSLR1* in rice. (**A**) Heat map of gene expression between WT vs. slr1-d7 and WT vs. slr1-d8 lines. Red denotes samples with relatively high expression of a given gene and blue denotes samples with relatively low expression. (**B**) Comparison of the number of differentially expressed genes (DEGs) in WT vs. slr1-d7 and WT vs. slr1-d8. (**C**) The number of DEGs up- and down-regulated between WT vs. mutant lines.

**Figure 4.** Gene ontology (GO) classification of DEGs shared by WT vs. slr1-d7 and WT vs. slr1-d8 lines. The *x*-axis shows user-selected GO terms, and the *y*-axis shows the enrichment ratio. Biological process: E, exocytosis; IPT, intracellular protein transport; MND, mitotic nuclear division; NST, nitric oxide mediated signal transduction; OT, oligopeptide transport; PHR, plant-type hypersensitive response; RK, response to karrikin; RST, response to salt stress; TTK, transmembrane receptor protein tyrosine kinase signaling pathway; VRM, vegetative to reproductive phase transition of meristem; NCP, nuclear-transcribed mRNA catabolic process. Cellular component: PM, plasma membrane; P, plasmodesma; GM, Golgi membrane; ER, endoplasmic reticulum; M, membrane; C, cytosol; ACP, anchored component of plasma membrane; GA, Golgi apparatus; ICM, integral component of membrane; TGN, trans-Golgi network; CM, chloroplast membrane; P, phragmoplast; CP, cytoplasmic mRNA processing body; PVM, plant-type vacuole membrane; VM, vacuolar membrane; E, endosome; LE, late endosome; EM, endosome membrane. Molecular function: TA, transporter activity; PKA, protein serine/threonine kinase activity; ATP, ATP binding; PB, protein binding; PTA, protein transporter activity; PA, potassium: proton antiporter activity; GDA, glucan endo-1,3-beta-D-glucosidase activity; SA, symporter activity; RA, ribonuclease activity.

**Figure 5.** Correlation analysis of gene expression pattern by RNA-Seq and qRT-PCR. ( **A**) WT vs. slr1-d7 line, (**B**) WT vs. slr1-d8 line.

#### *2.4. Key DEGs Related to Biosynthesis and Signaling Pathway of Plant Hormone*

In the RNA-seq analysis, the key DEGs related to plant hormone biosynthesis and 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, *ERF03*, *ERF110*, *BBM1* in ethylene biosynthesis, and *ILR1* in auxin biosynthesis. Additionally, 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, *ERF109*, *ERF39* in ethylene biosynthesis, *LOGL1* in cytokinin biosynthesis, *IAA7* in the auxin biosynthesis pathway, and *JAR1* in jasmonic acid (JA) biosynthesis (Supplementary Table S5). Among the DEGs associated with cell plate and leaf morphogenesis, Os05g0432200 and Os04g0407800 seemed to be important for the di fference between WT and edited lines. The expression levels of two GA-related genes, especially *GA20OX2* (gibberellin oxidase) and *GA3OX2*, were increased in the edited mutant plants compare to WT (Figure 5). The edited mutant lines are required by altering GA responses, at least partially by a defect in the phytohormone signaling process and prevented cell elongation.
