Different knockout genotypes of OsIAA23 in rice using CRISPR/Cas9 generating different phenotypes

Abstract

We have isolated several Osiaa23 rice mutants with different knockout genotypes, resulting in different phenotypes, which suggested that different genetic backgrounds or mutation types influence gene function. The Auxin/Indole-3-Acetic Acid (Aux/IAA) gene family performs critical roles in auxin signal transduction in plants. In rice, the gene OsIAA23 (Os06t0597000) is known to affect development of roots and shoots, but previous knockouts in OsIAA23 have been sterile and difficult for research continuously. Here, we isolate new Osiaa23 mutants using the CRISPR/Cas9 system in japonica (Wuyunjing24) and indica (Kasalath) rice, with extensive genome re-sequencing to confirm the absence of off-target effects. In Kasalath, mutants with a 13-amino acid deletion showed profoundly greater dwarfing, lateral root developmental disorder, and fertility deficiency, relative to mutants with a single amino acid deletion, demonstrating that those 13 amino acids in Kasalath are essential to gene function. In Wuyunjing24, we predicted that mutants with a single base-pair frameshift insertion would experience premature termination and strong phenotypic defects, but instead these lines exhibited negligible phenotypic difference and normal fertility. Through RNA-seq, we show here that new mosaic transcripts of OsIAA23 were produced de novo, which circumvented the premature termination and thereby preserved the wild-type phenotype. This finding is a notable demonstration in plants that mutants can mask loss of function CRISPR/Cas9 editing of the target gene through de novo changes in alternative splicing.

Keywords: Alternative splicing; CRISPR/Cas9; Oryza sativa; OsIAA23; Root development.

Fig. 1

Nucleotide diversity (π) and ratios of nonsynonymous to synonymous nucleotide substitutions (Ka/Ks) of IAA genes among rice genomes, including 166 indica rice, 167 japonica rice and 60 wild rice

Fig. 2

Targeted mutagenesis of OsIAA23 in rice using CRISPR/Cas system. a Diagram of gene structure and conserved domains of OsIAA23 (Os06t0597000-01 for representation). The black rectangles represent the exon regions. The short lines are marked with Roman numerals bellow indicate four domains of OsIAA23, and the spacer location is marked by red. b sgRNA:Cas9-induced rice OsIAA23 gene mutations at the target site in the mutants. The sequence marked in the red text under the black line indicates the spacer sequence. c Amino acid sequence alignment of wild type OsIAA23 protein and the Osiaa23 mutants. The sequence marked in the red text indicated the spacer location, and Domain II is indicated by underlined text

Fig. 3

Phenotypes of 7-day-old seedlings of wild type plants (WT), mutants of Osiaa23. Phenotype of 7-day-old seedlings of wild-type and mutants of Kasalath (a) and Wuyunjing24 (b), Bar = 2.5 cm. As marked, from left to right: wild-type (WT), the heterozygous mutants, and the homozygous mutants. Root phenotype of 7-day-old seedlings of wild-type and mutants of Kasalath (c) and Wuyunjing24 (d), Diameter = 1.4 cm. Enlarge views of root of the heterozygous mutants of Kasalath (e) and Wuyunjing24 (f)

Fig. 4

Growth parameters of wild type plants (WT), mutants of Osiaa23. The phenotypes of Osiaa23 mutants are compared with WT in the aspects of shoot length (a), crown root number (b), lateral root number (c), primary root length (d), crown root length (e), total root (fresh) weight (f), tiller number (g), mature plant height (h) and seed setting rate (i). Black columns represent Kasalath mutants, and gray columns represent Wuyunjing24 mutants. As marked at the bottom, from left to right: wild-type plants (WT), the heterozygous mutants, and the homozygous mutants. The standard deviation of the mean was denoted as error bar for each column. *p < 0.05, **p < 0.01 (Student’s t test)

Fig. 5

Phenotypes of mature plants of wild type (WT) and mutants of Osiaa23. Phenotype of plants at full ripening stage of wild-type (WT) and mutants of Kasalath (a) and Wuyunjing24 (b), Bar = 20 cm. Phenotypes of spikelet of wild-type (WT), mutants of Kasalath (c) and Wuyunjing24 (d). As marked at the bottom, from left to right: wild-type (WT), the heterozygous mutants, and the homozygous mutants

Fig. 6

Expression models of OsIAA23 and its homologs. Real-time PCR expression profiles of OsIAA23 gene in different tissues of wild-type (Ka-WT) and Wuyunjing24 (Wu-WT) (a). Comparison of expression levels of OsIAA23, OsIAA1 and OsIAA15 in the root of different mutants (b). The predicted transcripts, protein sequences and structures of OsIAA23 gene and edited Osiaa23 gene (c). The red vertical line marks the position of the gene editing. The amino acid sequences highlighted with the same color are the same. The sequences of Domain I, II, III and IV are indicated orderly by underline text. The standard deviation of the mean was denoted as error bar for each column. *p < 0.05, **p < 0.01