CRISPR/Cas9-mediated disruption of TaNP1 genes results in complete male sterility in bread wheat
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Abstract: Male sterile genes and mutants are valuable resources in hybrid seed production for monoclinous crops. High genetic redundancy due to allohexaploidy makes it difficult to obtain the nuclear recessive male sterile mutants through spontaneous mutation or chemical or physical mutagenesis methods in wheat. The emerging effective genome editing tool, CRISPR/Cas9 system, makes it possible to achieve simultaneous mutagenesis in multiple homoeoalleles. To improve the genome modification efficiency of the CRISPR/Cas9 system in wheat, we compared four different RNA polymerase (Pol) III promoters (TaU3p, TaU6p, OsU3p, and OsU6p) and three types of sgRNA scaffold in the protoplast system. We show that theTaU3 promoter-driven optimized sgRNA scaffold was most effective. The optimized CRISPR/Cas9 system was used to edit three TaNP1 homoeoalleles, whose orthologs, OsNP1 in rice and ZmIPE1 in maize, encode a putative glucose-methanol-choline oxidoreductase and are required for male sterility. Triple homozygous mutations in TaNP1 genes result in complete male sterility. We further demonstrated that any one wild-type copy of the three TaNP1 genes is sufficient for maintenance of male fertility. Taken together, this study provides an optimized CRISPR/Cas9 vector for wheat genome editing and a complete male sterile mutant for development of a commercially viable hybrid wheat seed production system.
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Key words:
- Wheat /
- CRISPR/Cas9 /
- Male sterility
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Fig. 1. Protein sequence alignment of TaNP1 orthologs across grass species. A: The protein sequence alignment of three TaNP1 genes and their orthologs in other grass species using software BioEdit (7.0.9.0). OsNP1, XP_015614422.1; ZmIPE1, ONL99356.1; TaNP1-A1, MT093833; TaNP-B1, MT108234; TaNP1-D1, MT108235; HvNP1, BAK07510.1; BdNP1, XP_003574225.1. B: Protein sequence identity between each other using Clustal Omega (http://www.clustal.org/omega/).
Fig. 2. Expression pattern of TaNP1 genes. A: Expression analysis of three TaNP1 genes by RT-PCR (A, upper) and quantitative RT-PCR (A, lower) in different tissues of wheat. Le&pa, lemma and palea; UM, unicellular microspore stage; BP, bicellular pollen stage; MP, mature pollen stage.ACTIN was used as an internal control. Data are shown as means ± SEM (n = 3). B: In situ hybridization analysis of TaNP1 in anthers of wheat. Sense TaNP1 probe was used as control. Dy, dyad cell; MC, meiotic cell; Msp, microspore; T, tapetum; Tds, tetrads. Bar = 50 μm.
Fig. 3. Optimization of the CRISPR/Cas9 system and target selection for TaNP1 genes in wheat protoplasts. A–C: PCR/RE assay to detect mutations in TaNP1 genes (A, B, D genome) induced by different CRISPR/Cas9 vectors in wheat protoplasts, respectively. A: The mutation rates induced by four different CRISPR/Cas9 vectors, whose sgRNA scaffolds are driven by four different Pol III promoters, were compared. The sequence of target site (TS2) is from a conserved region of threeTaNP1 genes. The BanI restriction site is underlined. B: The mutation rates induced by three different CRISPR/Cas9 vectors were compared. pGR vector contains the most commonly used sgRNA scaffold, pOPGR vector contains the optimized sgRNA scaffold, and pTRGR vector contains a structure of tRNAGly-sgRNA. C: Six target sites sequences were constructed into pOPGR vector. And the mutation rates induced by the six CRISPR/Cas9 vectors were compared. Lane 1, digested PCR amplicons from CRISPR/Cas9 vector transformed protoplasts. Lane 2 and 3, digested and undigested PCR amplicons from WT. TS, target site. Restriction enzyme BanI was used for TS1, TS2, and TS6; BsaHI was used for TS3; DdeI was used for TS4; StyI was used for TS5. Red arrows indicate the mutation bands.
Fig. 4. Targeted mutation of TaNP1 genes using the CRISPR/Cas9 system. A: PCR/RE assay to detect CRISPR/Cas9-induced mutations at TS5 target of TaNP1 genes in 12 representative T0 transgenic wheat plants. Lanes T0-1 to T0-12 show PCR products amplified from transgenic plants with specific primers and digested with StyI. Lanes labeled WT show PCR products amplified from a wild-type control plant with or without StyI digestion. Red arrows indicate the mutation bands. B: Representative mutations in TaNP1 genes from T0 transgenic plants. Green letters indicate the PAM sequences. Restriction enzyme sites are underlined. Deletions and insertions are indicated by dashes and blue letters, respectively. Numbers on the right side indicate types of mutation and numbers of nucleotides involved.
Fig. 5. Morphological comparison between the wild-type and tanp1 mutants at TS5. A: Spikes of wild-type and tanp1 mutants. Bar = 1 cm. B and C: Anthers of wild-type and tanp1 mutants before (B) and after filament elongation (C). Bar = 1 mm. D: Mature pollen grains of wild-type and tanp1 stained with I2-KI. Bar = 200 μm. E: Seeds of wild-type and tanp1 at 20 days after pollination. Bar = 1 mm.
Table 1. Genotypes and seed set for T1 progenies of T0-8.
Genotype Seed set TaNP-A1 TaNP-B1 TaNP-D1 Plants Total seeds Seeds per plant a2a2 b1b1 d1d1 2 0 0 a1a2 b1b1 d2d2 2 0 0 a2a2 b1b1 d1d2 1 0 0 a1a2 b1b1 d1d2 2 0 0 a1a1 b2b2 d1d1 2 252 126 a1a2 b2b2 d1d1 2 266 133 a2a2 b2b2 d1d2 3 448 149 a1a1 b2b2 d1d2 1 27 27 a1a2 b1b2 d1d1 3 406 135 a1a2 b1b2 d2d2 2 232 116 a1a1 b1b2 d2d2 1 38 38 a2a2 b1b2 d2d2 1 286 286 a1a2 b1b2 d1d2 6 598 100 a1a1 b1b2 d1d2 2 107 54 a2a2 b1b2 d1d2 2 268 134 WT WT WT 5a 1130 226 WT, wild-type. aThese plants are wild-type, which are not the progenies of T0-8. 
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