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. 2021 Apr;19(4):645-647.
doi: 10.1111/pbi.13515. Epub 2020 Dec 13.

A CRISPR/Cas9-based genome-editing system for yam (Dioscorea spp.)

Easter D Syombua  1   2 , Zhengzhi Zhang  3 , Jaindra N Tripathi  1 , Valentine O Ntui  1 , Minjeong Kang  4 , Obiero O George  2 , Nguu K Edward  5 , Kan Wang  4 , Bing Yang  3   6 , Leena Tripathi  1
Affiliations

A CRISPR/Cas9-based genome-editing system for yam (Dioscorea spp.)

Easter D Syombua et al. Plant Biotechnol J. 2021 Apr.
No abstract available

Keywords: DrPDS; CRISPR/Cas9; Dioscorea spp.; Yam; genome-editing.

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Conflict of interest statement

Authors declare no conflict of interest.

Figures

Figure 1
Figure 1
CRISPR/Cas9‐mediated genome‐editing of yam targeting phytoene desaturase (DrPDS) gene. (a) Expression of GFP in protoplasts transfected with pCas9‐DaU6::gGFP + 1 and non‐functional p35S::GFP + 1. Scale bars, 25 µm. (b) Editing efficiency of Cas9 and DaU6 promoters in yam protoplasts. Fluorescence was scored as scale from 1 to 10, with no tracked fluorescence scored as 0 for p35S::GFP + 1, scored as 10 for pUbi::GFP. (c) Gene structure of DrPDS showing the gRNA targets. Black bars indicate exons of the gene, PAM sequences in blue and underlined, and protospacer sequences in red. (d) Schematic presentation of pCas9_gRNA‐PDS used to generate genome‐edited events. (e) Transient gene expression in yam leaves agroinfiltrated with Agrobacterium harbouring pCas9_gRNA‐PDS. (e‐1) Leaf infiltrated with infection medium only, (e‐2&e‐3) leaf infiltrated with Agrobacterium showing bleached patches, (e‐4) microscopic examination of an infiltrated leaf section, (e‐5) green fluorescent micrograph of the infiltrated section, (e‐6) green fluorescent micrograph of an infiltrated section heat treated at 2 dpi and photographed at 4 dpi. Scale bar: e‐1, e‐2 and e‐3, 1 cm, e‐4, e‐5, and e‐6, 0.25 mm. (f) Genome‐edited events of yam accession Amola. (f‐1) Albino shoot (#5) on the selective medium, (f‐2) Albino shoots (#1) with a bushy phenotype, (f‐3) complete albino (left, #5 ) and wild‐type (right, #7) plantlet, (f‐4) variegated albino plantlets (#6&8), (f‐5) genome‐edited leaf of #5 under UV light, (f‐6) wild‐type leaf under UV light. Scale bar: f‐1, f‐2 and f‐3, 1 cm; f‐4, f‐5, and f‐6, 0.25 mm. (g) Site‐specific mutations of DrPDS induced by two gRNAs. PAM is in blue and protospacers in red. Deletions are denoted by black dashes and insertion by green.
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References

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