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. 2020 Apr;18(4):889-891.
doi: 10.1111/pbi.13264. Epub 2019 Oct 7.

High-efficiency CRISPR/Cas-based editing of Phalaenopsis orchid MADS genes

Chii-Gong Tong #  1 , Fu-Hui Wu #  1 , Yu-Hsuan Yuan #  1 , Yan-Ru Chen  1 , Choun-Sea Lin  1
Affiliations

High-efficiency CRISPR/Cas-based editing of Phalaenopsis orchid MADS genes

Chii-Gong Tong et al. Plant Biotechnol J. 2020 Apr.
No abstract available

Keywords: agrobacterium-mediated transformation; gene family; protospacer-adjacent motif; transformation strategy.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Strategies and results of CRISPR/Cas targeted mutagenesis of Phalaenopsis MADS genes. Homozygous, harbours the same edited sequences in both alleles; biallelic, both alleles were edited but the sequences were different; chimera, more than two alleles in the transformants; heterozygous, one wild type and one edited allele. In (g) and (j), letters in red, mutation; – in red, deletion. Numbers in parentheses indicate the number of bases deleted; ‘Hetero’, heterozygous. The coloured blocks in (h) and (k) indicate the genotypes of transformants that were edited in the genes shown above the column. Red, homozygous; orange, biallelic; yellow, chimera; purple, heterozygous and green, mutated in the sequences in MADS44 that are similar to the MADS8 target site. (a). One‐month‐old protocorms were incubated with 3sg1C Agrobacteria. Bar = 1 cm. (b). Transfected protocorms were incubated in hygromycin medium. The green protocorms are putative transformed explants. Bar = 1 cm. (c). Transformants with green true leaves. Bar = 1 cm. (d). Rooted 3sg1C#17‐1 to ‐4 incubated in hygromycin medium after 1‐month of subculture. Bar = 1 cm. (e). Two‐month‐old 3sg1C#13‐1 transformant. Bar = 0.5 cm. (f). In the 3sg1C#8‐3 transformant, three MADS gene target‐site regions were amplified and sequenced. Blue bar, target site; red bar, protospacer‐adjacent motif (PAM). Multiple peaks start from the sequences near the PAM indicating that there were mutated PCR products. (g). 3sg1C#8‐3 PCR products of three MADS gene target‐site regions were cloned, and eight clones from each construct were sequenced to determine the genotype. (h). MADS8, MADS36 and MADS44 target gene analysis in 3sg1C transformants. The explants are distinguished by lines (#1 to #21). Each row indicates one transformant (‐1 to ‐4). (i). DNA was isolated from each transformant derived from the 3X1sg strategy for PCR, including the target regions of MADS genes ( MADS8, MADS36 and MADS44); sgRNAs of each construct (sgRNA8, sgRNA36 and sgRNA44); Cas9 and actin as an internal control. The MADS gene PCR products were combined with wild‐type MADS DNA and tested using a T7 Endonuclease I assay. Cleavage of the PCR product indicates the presence of a mutation in this transformant. (j). These mutated MADS gene PCR products in (i) were cloned, and eight clones were sequenced for each construct. (k). MADS8, MADS36 and MADS44 target gene and sgRNA analysis in 3X1sg transformants. The coloured blocks indicate the genotype of transformants that were edited in the genes shown above the column.
See this image and copyright information in PMC

References

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