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. 2022 Jan 27;11(3):439.
doi: 10.3390/cells11030439.

Use of CRISPR/Cas9-Based Gene Editing to Simultaneously Mutate Multiple Homologous Genes Required for Pollen Development and Male Fertility in Maize

Xinze Liu  1 , Shaowei Zhang  1 , Yilin Jiang  1 , Tingwei Yan  1 , Chaowei Fang  1 , Quancan Hou  1   2 , Suowei Wu  1   2 , Ke Xie  1   2 , Xueli An  1   2 , Xiangyuan Wan  1   2
Affiliations

Use of CRISPR/Cas9-Based Gene Editing to Simultaneously Mutate Multiple Homologous Genes Required for Pollen Development and Male Fertility in Maize

Xinze Liu et al. Cells. .

Abstract

Male sterility represents an important trait for hybrid breeding and seed production in crops. Although the genes required for male fertility have been widely studied and characterized in many plant species, most of them are single genic male-sterility (GMS) genes. To investigate the role of multiple homologous genes in anther and pollen developments of maize, we established the CRISPR/Cas9-based gene editing method to simultaneously mutate the homologs in several putative GMS gene families. By using the integrated strategies of multi-gene editing vectors, maize genetic transformation, mutation-site analysis of T0 and F1 plants, and genotyping and phenotyping of F2 progenies, we further confirmed gene functions of every member in ZmTGA9-1/-2/-3 family, and identified the functions of ZmDFR1, ZmDFR2, ZmACOS5-1, and ZmACOS5-2 in controlling maize male fertility. Single and double homozygous gene mutants of ZmTGA9-1/-2/-3 did not affect anther and pollen development, while triple homozygous gene mutant resulted in complete male sterility. Two single-gene mutants of ZmDFR1/2 displayed partial male sterility, but the double-gene mutant showed complete male sterility. Additionally, only the ZmACOS5-2 single gene was required for anther and pollen development, while ZmACOS5-1 had no effect on male fertility. Our results show that the CRISPR/Cas9 gene editing system is a highly efficient and convenient tool for identifying multiple homologous GMS genes. These findings enrich GMS genes and mutant resources for breeding of maize GMS lines and promote deep understanding of the gene family underlying pollen development and male fertility in maize.

Keywords: CRISPR/Cas9; gene editing; genic male sterility; maize; multiple homologous genes; pollen development.

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Conflict of interest statement

The authors declare no conflict of interest. Beijing Solidwill Sci-Tech Co., Ltd., the Beijing Engineering Laboratory, and Beijing International Science and Technology Cooperation Base provided the research platform, instruments, and funds for this study; thus, the company is one of the signed units of this paper.

Figures

Figure 1
Figure 1
The CRISPR/Cas9 mutagenesis of ZmTGA9-1, ZmTGA9-2, and ZmTGA9-3 and the derived mutant sequencing analysis. (A) The CRISPR/Cas9 mutagenesis of ZmTGA9-1, ZmTGA9-2, and ZmTGA9-3. (A1) Diagram showing the two target sites on the ZmTGA9-1, ZmTGA9-2, and ZmTGA9-3, respectively. (A2) Sequence analysis of the target sites on ZmTGA9-1, ZmTGA9-2, and ZmTGA9-3 and their mutants. The target sites and protospacer-adjacent motif (PAM) sequences are shown in the antisense strand and highlighted in red and boldface fonts, respectively. Deletions and insertions are indicated by red, short, dashed lines and green fonts, respectively. (A3) Diagram illustrating the sgRNA expression cassettes targeting ZmTGA9-1, ZmTGA9-2, and ZmTGA9-3 via the dual-sgRNAs CRISPR/Cas9 vector system. (B) Identification of mutations in a homozygous zmtga9-1/-2/-3 triple knockout line by Sanger sequencing. (B1) Sanger sequencing result of mutation in the ZmTGA9-1 gene. (B2) Sanger sequencing result of mutation in the ZmTGA9-2 gene. (B3) Sanger sequencing result of mutation in the ZmTGA9-3 gene. Deletions and insertions are indicated by red, short, dashed lines and green fonts, respectively.
Figure 2
Figure 2
Co-segregating molecular marker genotyping and phenotypic characterization of ZmTGA9-1/-2/-3-Cas9 mutants in F2 population. (A) The genotypic identifications of representative ZmTGA9-1/-2/-3-Cas9 F2 individuals using developed co-segregating molecular markers. Molecular markers covering 1-bp deletion, 1-bp insertion and 1-bp insertion in the ZmTGA9-1/-2/-3 genes were detected by using PCR amplification with PAGE, respectively. M, marker. (B) Phenotypic analysis of tassels, anthers and pollen grains stained with 1% I2-KI solution in WT and the single-, double- and triple-gene homozygous mutants of ZmTGA9-1/-2/-3. A and a, B and b, and C and c represent the WT and mutant genotypes of ZmTGA9-1/-2/-3, respectively. A_/B_/C_ consists of AA/BB/CC, Aa/BB/CC, AA/Bb/CC, AA/BB/Cc, Aa/Bb/CC, Aa/BB/Cc, AA/Bb/Cc, and Aa/Bb/Cc genotypes. aa/B_/C_ consists of aa/BB/CC, aa/Bb/CC, aa/BB/Cc, and aa/Bb/Cc genotypes. A_/bb/C_ consists of AA/bb/CC, Aa/bb/CC, AA/bb/Cc, and Aa/bb/Cc genotypes. A_/B_/cc consists of AA/BB/cc, Aa/BB/cc, AA/Bb/cc, and Aa/Bb/cc genotypes. aa/bb/C_ consists of aa/bb/CC and aa/bb/Cc genotypes. aa/B_/cc consists of aa/BB/cc and aa/Bb/cc genotypes. A_/bb/cc consists of AA/bb/cc and Aa/bb/cc genotypes.
Figure 3
Figure 3
The CRISPR/Cas9 mutagenesis of ZmDFR1 and ZmDFR2, and co-segregating molecular marker genotyping and phenotypic characterization of ZmDFR1/2-Cas9 mutants in F2 population. (A) The CRISPR/Cas9 mutagenesis of ZmDFR1 and ZmDFR2. (A1) Diagram showing the two target sites on the ZmDFR1 and ZmDFR2 genes, respectively. (A2) Sequence analysis of the target sites in on ZmDFR1 and ZmDFR2 and their mutants. The target sites and PAM sequences are shown in the sense strand and highlighted in red and boldface fonts, respectively. Deletions are indicated by red, short, dashed lines. (A3) Diagram illustrating the sgRNA expression cassettes targeting ZmDFR1 and ZmDFR2 genes via the dual-sgRNAs CRISPR/Cas9 vector system. (B) Identification of mutations in a homozygous zmdfr1/2 double knockout line by Sanger sequencing. (B1) Sanger sequencing result of mutation in ZmDFR1 gene. (B2) Sanger sequencing result of mutation in the ZmDFR2 gene. Deletions are indicated by red, short, dashed lines. (C) The genotypic identifications of representative ZmDFR1/2-Cas9 F2 individuals using developed co-segregating molecular markers. Molecular markers covering 1-bp deletion and 1-bp deletion in ZmDFR1 and ZmDFR2 genes were detected by using PCR amplification with PAGE, respectively. M, marker. (D) Phenotypic characterization of ZmDFR1/2-Cas9 mutants in F2 population. (D1) Phenotypic analysis of tassels, anthers and pollen grains stained with 1% I2-KI solution in WT and the single- and double-gene homozygous mutants of ZmDFR1/2. (D2) The proportions of normal and aborted pollen grains measured by staining with 1% I2-KI solution in WT and the single-gene mutants of ZmDFR1 and ZmDFR2 at stage 13 (n = 4220 to 4300). A and a represent the WT and mutant genotypes of ZmDFR1, respectively. B and b represent the WT and mutant genotypes of ZmDFR2, respectively. A_/B_ consists of AA/BB, Aa/BB, AA/Bb, and Aa/Bb genotypes. aa/B_ consists of aa/BB and aa/Bb genotypes. A_/bb consists of AA/bb and Aa/bb genotypes.
Figure 4
Figure 4
The CRISPR/Cas9 mutagenesis of ZmACOS5-1 and ZmACOS5-2, and co-segregating molecular marker genotyping and phenotypic characterization of ZmACOS5-1/-2-Cas9 mutants in F2 population. (A) The CRISPR/Cas9 mutagenesis of ZmACOS5-1 and ZmACOS5-2. (A1) Diagram showing the two target sites on the ZmACOS5-1 and ZmACOS5-2 genes, respectively. (A2) Sequence analysis of the target sites in on ZmACOS5-1 and ZmACOS5-2 and their mutants. The target sites and PAM sequences are shown in the sense strand and highlighted in red and boldface fonts, respectively. Insertions are indicated by green fonts. The sequence gap length is shown in the middle of the sequences. (A3) Diagram illustrating the sgRNA expression cassettes targeting ZmACOS5-1 and ZmACOS5-2 genes via the dual-sgRNAs CRISPR/Cas9 vector system. (B) Identification of mutations in a homozygous zmacos5-1/-2 double knockout line by Sanger sequencing. (B1) Sanger sequencing result of mutation in the ZmACOS5-1 gene. (B2) Sanger sequencing result of mutation in the ZmACOS5-2 gene. Deletions are indicated by red, short, dashed lines. (C) The genotypic identifications of representative ZmACOS5-1/-2-Cas9 F2 individuals using developed co-segregating molecular markers. Molecular markers covering 1-bp insertion and 1-bp insertion in ZmACOS5-1 and ZmACOS5-2 genes were detected by using PCR amplification with PAGE, respectively. (D) Phenotypic analysis of tassels, anthers and pollen grains stained with 1% I2-KI solution in WT and the single- and double-gene homozygous mutants of ZmACOS5-1/-2. A and a represent the WT and mutant genotypes of ZmACOS5-1, respectively. B and b represent the WT and mutant genotypes of ZmACOS5-2, respectively. A_/B_ consists of AA/BB, Aa/BB, AA/Bb, and Aa/Bb genotypes. Aa/B_ consists of aa/BB and aa/Bb genotypes. A_/bb consists of AA/bb and Aa/bb genotypes.
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