CRISPR/Cas9 Directed Mutagenesis of OsGA20ox2 in High Yielding Basmati Rice (Oryza sativa L.) Line and Comparative Proteome Profiling of Unveiled Changes Triggered by Mutations

Abstract

In rice, semi-dwarfism is among the most required characteristics, as it facilitates better yields and offers lodging resistance. Here, semi-dwarf rice lines lacking any residual transgene-DNA and off-target effects were generated through CRISPR/Cas9-guided mutagenesis of the OsGA20ox2 gene in a high yielding Basmati rice line, and the isobaric tags for relative and absolute quantification (iTRAQ) strategy was utilized to elucidate the proteomic changes in mutants. The results indicated the reduced gibberellins (GA₁ and GA₄) levels, plant height (28.72%), and flag leaf length, while all the other traits remained unchanged. The OsGA20ox2 expression was highly suppressed, and the mutants exhibited decreased cell length, width, and restored their plant height by exogenous GA₃ treatment. Comparative proteomics of the wild-type and homozygous mutant line (GXU43_9) showed an altered level of 588 proteins, 273 upregulated and 315 downregulated, respectively. The identified differentially expressed proteins (DEPs) were mainly enriched in the carbon metabolism and fixation, glycolysis/gluconeogenesis, photosynthesis, and oxidative phosphorylation pathways. The proteins (Q6AWY7, Q6AWY2, Q9FRG8, Q6EPP9, Q6AWX8) associated with growth-regulating factors (GRF2, GRF7, GRF9, GRF10, and GRF11) and GA (Q8RZ73, Q9AS97, Q69VG1, Q8LNJ6, Q0JH50, and Q5MQ85) were downregulated, while the abscisic stress-ripening protein 5 (ASR5) and abscisic acid receptor (PYL5) were upregulated in mutant lines. We integrated CRISPR/Cas9 with proteomic screening as the most reliable strategy for rapid assessment of the CRISPR experiments outcomes.

Keywords: CRISPR/Cas9; OsGA20ox2; gibberellins; plant height; proteomic analysis; rice.

Figure 1

Identification of mutations generated by the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. (A) The mutation efficiency of sgRNAs (single guided RNAs), (B) The mutation rate in T₀ (Transgenic) generation, (C) sequencing chromatograms of WT (wild type) and homozygous mutant lines for Target 1 and Target 2, and (D) the alignment of sequences for T₁, T₂, and T₃ generations, respectively. The target sequence is painted in yellow, while the PAM (protospacer adjacent motif) is in the green background, and insertions/deletions are represented by red hyphens and letters. The analysis was carried out in three replications for each line.

Figure 1

Identification of mutations generated by the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. (A) The mutation efficiency of sgRNAs (single guided RNAs), (B) The mutation rate in T₀ (Transgenic) generation, (C) sequencing chromatograms of WT (wild type) and homozygous mutant lines for Target 1 and Target 2, and (D) the alignment of sequences for T₁, T₂, and T₃ generations, respectively. The target sequence is painted in yellow, while the PAM (protospacer adjacent motif) is in the green background, and insertions/deletions are represented by red hyphens and letters. The analysis was carried out in three replications for each line.

Figure 2

Phenotypic appearance of OsGA20ox2 in GXU43_9 and WT. (A) Plant height of mutant lines and WT after the heading stage. Bar = 15 cm. (B) Grain phenotype of mutant line and WT; Bar = 5 mm. (C) The lengths of internodes in mutant line and WT. The “*” denotes the significant difference at p < 0.01. Values are means ± standard deviation (SD) (n = 10 plants).

Figure 3

The seedling phenotype of the homozygous mutant GXU43_9 and WT. (A) Seedlings phenotype without GA, (B) Seedlings treated with GA, and (C) PH (plant height)of GA3-treated and control (n = 15), Bars = 3 cm. Data are mean ± SD. “**” and “^ns” represent a significant and non-significant difference respectively at p ≤ 0.01.

Figure 4

The microscopic analysis of culm cells of WT and mutant line GXU43_9. (A) WT second intermodal longitudinal length of the cell and (B) mutant plant GXU43_9, respectively. (C,D) Quantitative measurement of the length and width of the cells of mutant line (GXU43_9) and wild-type plants (n = 15), Bars = 100 μm. Data are the mean ± SD, “**” shows the significant difference at p ≤ 0.01.

Figure 5

Proteomic analysis information of the CRISPR mutant GXU43_9 and its WT. (A) Upregulated and downregulated differentially expressed proteins (DEPs), and (B) Heatmap of the top twenty DEPs. Red color denotes the higher while the green color represents a lower level of expression.

Figure 6

Functional networks of DEPs and hub-protein analysis of WT and mutant line GXU43_9. (A) STRING software predicted proteins’ associations. The nodes represent differentially expressed proteins, while the edge denotes the interaction relationship between the nodes. (B) The top hub-proteins of WT and GXU43_9 comparison. The higher co-expression is denoted by red color.

Figure 7

Gene ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis of DEPs. (A) GO annotations of the DEPs, and (B) the histogram of KEGG pathway enrichment, with the bar showing the number of proteins. BP; biological processes, CC; cellular components, and MF; molecular functions.

Figure 8

Real-time quantitative PCR assessment and validation of proteomics data. (A) Expression analysis of OsGA20ox2, in WT and T₁ mutant lines, (B) RT qPCR validation of the ten DEPs responsive genes. The SD is shown with error bars, “*” represents the significant differences at p ≤ 0.05 and “**” represents the significant differences at p ≤ 0.01 respectively; n = 3.