Efficient Multi-Sites Genome Editing and Plant Regeneration via Somatic Embryogenesis in Picea glauca

Abstract

Conifers are the world's major source of timber and pulpwood and have great economic and ecological value. Currently, little research on the application of CRISPR/Cas9, the commonly used genome-editing tool in angiosperms, has been reported in coniferous species. An efficient CRISPR/Cas9 system based on somatic embryogenesis (SEis) suitable for conifers could benefit both fundamental and applied research in these species. In this study, the SpCas9 gene was optimized based on codon bias in white spruce, and a spruce U6 promoter was cloned and function-validated for use in a conifer specific CRISPR/Cas9 toolbox, i.e., PgCas9/PaU6. With this toolbox, a genome-editing vector was constructed to target the DXS1 gene of white spruce. By Agrobacterium-mediated transformation, the genome-editing vector was then transferred into embryogenic tissue of white spruce. Three resistant embryogenic tissues were obtained and used for regenerating plants via SEis. Albino somatic embryo (SE) plants with mutations in DXS1 were obtained in all of the three events, and the ratios of the homozygous and biallelic mutants in the 18 albino mutants detected were 22.2% in both cases. Green plants with mutations in DXS1 were also produced, and the ratios of the DXS1 mutants to the total green plants were 7.9, 28, and 13.5%, respectively, among the three events. Since 22.7% of the total 44 mutants were edited at both of the target sites 1 and 2, the CRISPR/Cas9 toolbox in this research could be used for multi-sites genome editing. More than 2,000 SE plants were regenerated in vitro after genome editing, and part of them showed differences in plant development. Both chimerism and mosaicism were found in the SE plants of white spruce after genome editing with the CRISPR/Cas9 toolbox. The conifer-specific CRISPR/Cas9 system developed in this research could be valuable in gene function research and trait improvement.

Keywords: CRISPR/Cas9; DXS1; Picea glauca; conifer; genome editing; gymnosperm; plant regeneration; somatic embryogenesis.

Figure 1

The T-DNA structure of PgCas9/PaU6. The hygromycin phosphate transferase (hpt) gene, which was driven and terminated by the CaMV35S promoter and the CaMV35S polyA, respectively, was used as the selective marker gene. The PgCas9 with a nuclear localization signal (NLS) at each side of 5′ and 3′ ends was driven by the doubled 35S promoter and terminated by the Nos terminator. The PaU6 promoter was used to drive the expression of polycistronic tRNA-gRNA (PTG), and two BsaI sites were designed between the PaU6 promoter and the gRNA backbone for assembling the PTG into the binary vector. EcoRI, SacI, NcoI, BamHI, SalI, and HindIII were restriction enzymes. The left and right borders of T-DNA were designated as LB and RB, respectively.

Figure 2

Target sites in the DXS1 gene and regeneration of transgenic somatic embryo (SE) plants. The sequences of two target sites in the DXS1 gene and the primers used for the detection of mutations were shown in (A). Letters in italic bold type were PAM sites. The primers were shown under the DXS1 gene with their names and positions indicated. Hygromycin-resistant embryogenic tissue was obtained about 4 weeks after transformation (B). Resistant embryogenic tissues proliferated quickly on the selection medium (C). Cotyledonary SEs developed after 4 weeks of maturation (D) and further converted into SE plants on the germination medium (E–H). (E,F) showed the germination of green and albino SE plants, respectively. (G,H) showed the growth of albino and mosaic SE plants after 20 days on the germination medium, which was indicated with red arrows. (I) showed the results of PCR amplification of hpt gene in the transgenic SEs (1–20), wild-type SEs (WT), and the positive control PgCas9/PaU6 (DXS1) (P). SE plants of wild type and transgenic types, including green, albino, and mosaic ones after 65 days on the germination medium, were shown in (J–M), respectively. All bars equal to 500 μm in (C–H), while bars in (J–M) equal to 1,000 μm.

Figure 3

Sequences at the target sites in albino SE plants. Sequencing chromatograms of biallelic (2–44, 2–49, 3–43, and 3–45) and homozygous (2–43, 2–48, 3–16, and 3–42) mutations were shown in (A). The red arrows indicated the sites of mutations. The edited sequences in the 18 albino SE plants were listed in (B). The sequences at the target site 1 and the target site 2 in wild-type SE plants of WSP3 were underlined. PAM sites were indicated with red color. The line number of the individual transgenic line was indicated on the left side of the sequence. The nucleotide insertion and deletion were indicated by +n bp and -n bp, respectively, on the right side of the sequence.

Figure 4

The process of somatic embryogenesis (SEis) and genome editing in Picea glauca. The procedure of developing a SEis system and genome editing with the desired embryogenic cell line is shown in this diagram. ET, SEis, SEs, and PTGs stand for embryogenic tissue, somatic embryogenesis, somatic embryos, and polycistronic tRNA-gRNAs, respectively.