Plant Biotechnology Journal (2020), pp. 1–3 doi: 10.1111/pbi.13515
Brief Communication
A CRISPR/Cas9-based genome-editing system for yam
(Dioscorea spp.)
Easter D. Syombua1,2, Zhengzhi Zhang3, Jaindra N. Tripathi1 , Valentine O. Ntui1, Minjeong Kang4,
Obiero O. George2, Nguu K. Edward5, Kan Wang4,* , Bing Yang3,6,* and Leena Tripathi1,*
1International Institute of Tropical Agriculture (IITA), Nairobi, Kenya
2Centre for Biotechnology and Bioinformatics, University of Nairobi, Nairobi, Kenya
3Division of Plant Sciences, Bond Life Sciences Center, University of Missouri, Columbia, MO, USA
4Department of Agronomy, Iowa State University, Ames, IA, USA
5Department of Biochemistry, University of Nairobi, Nairobi, Kenya
6Donald Danforth Plant Science Center, St. Louis, MO, USA
We first sought to identify the promoters for expressing guide
Received 23 September 2020; RNAs (gRNAs) in yam. Five U6 genes from D. alata were
revised 27 October 2020; identified, and the respective promoters (~300 bp) were synthe-
accepted 6 November 2020. sized. To identify the best DaU6 promoters, a gRNA targeting a
*Correspondence Leena Tripathi (Tel +254-20-422-3472; fax +254-20-422- mutated green fluorescence protein gene (GFP + 1) was con-
3001; L.Tripathi@cgiar.org), Kan Wang (Tel +1-515-294-4429; fax +1-515-294- structed under each DaU6 promoter. Individual gRNAs and Cas9
3163; kanwang@iastate.edu), Bing Yang (Tel +1-573-882-7705; fax +1573-
(pCas9-DaU6::gGFP + 1) and p35S::GFP + 1 were mixed equally
884-9676; yangbi@missouri.edu)
and introduced into the yam mesophyll protoplasts, while pUbi::
Keywords: Yam, Dioscorea spp., genome-editing, CRISPR/Cas9, GFP and p35::GFP + 1 were used as positive and negative
control, respectively, using the method described previously for
DrPDS.
assessing the efficacy of wheat U6 promoters (Zhang et al.,
2019). Protoplasts transfected with pUbi::GFP showed strong
To Editor fluorescence 40 h post-transfection (Figure 1a). In contrast, no
Yam (Dioscorea spp.) is a multi-species tuber crop providing fluorescence was observed in protoplasts with non-functional
food and income to millions of people worldwide, particularly in GFP + 1. Some protoplasts transfected with pCas9-DaU6::
Africa (Price et al., 2016). The ‘yam belt’ in West Africa, including gGFP + 1 and p35S::GFP + 1 together showed GFP fluorescence,
Nigeria, Benin, Togo, Ghana, and Côte d’Ivoire, accounts for 92% indicating the GFP + 1 was correctly edited into the functional
of 72.6 million tons of global yam production (FAOSTAT, 2018). gfp gene (Figure 1a). A comparison of the efficacy of different
Despite the economic importance, yam cultivation is plagued by yam U6 promotors (DaU6.1 to DaU6.5) using the protoplast
several biotic and abiotic factors. Yam genetic improvement via transfection assay showed variation in the number of GFP-
conventional breeding has not achieved substantial progress fluorescing protoplasts and their intensity. Promoter DaU6.5
mainly due to the dioecy nature, long breeding-cycle, polyploidy, performed best, while DaU6.2 and DaU6.3 yielded similar
heterozygosity, poor seed set, and non-synchronous flowering fluorescence scores (Figure 1b). Consequently, we selected pro-
(Mignouna et al., 2008). A precise genome-engineering holds the moters DaU6.3 and DaU6.5 to direct the gRNA expression for
potential to overcome some of these limitations. CRISPR/Cas9 is stable transformation of yam. The complete sequence of DrPDS
the most popular genome-editing system applied extensively for was identified by Blast searching the NCBI database using
crop improvement, wherein yam is lagging far behind other crop Arabidopsis phytoene desaturase 3 protein (NP_193157.1) (Fig-
species. The genetic transformation technologies and genome ure 1c). A plasmid construct, pCas9-gRNA-PDS, was built. This
sequences, only recently available, made it possible to realize the construct carries a Cas9-gfp fusion gene driven by maize ubiquitin
potential of CRISPR-based genome editing for basic and applied promoter (Zhang et al., 2019), two gRNAs targeting exon 2 of
research in yam (Manoharan et al., 2016; Nyaboga et al., 2014; DrPDS (Figure 1c) under DaU6.3 and DaU6.5 promoters individ-
Tamiru et al., 2017). Here, we report, for the first time, the ually, and a plant selectable marker nptII gene under CaMV35S
successful establishment of a CRISPR/Cas9-based genome-editing promoter (Figure 1d).
system and validation of its efficacy by targeting the phytoene We next evaluated the efficacy of the Cas9-gfp gene expres-
desaturase gene (DrPDS) in a West African farmer-preferred D. sion in yam using agroinfiltration. The agroinfiltration-based
rotundata accession Amola. The PDS gene is involved in convert- system was established through the infiltration of young leaves of
ing phytoene into carotenoid precursors phytofluene and f- two months old potted plants with Agrobacterium harbouring
carotene (Mann et al., 1994). It is commonly used as a visual the construct pCas9-gRNA-PDS (Figure 1d). The effect of various
marker to validate genome editing in plants, as disruption of its factors, including the age of leaves (young, unopened; young,
function causes albinism. fully expanded; and mature), infiltration buffer, Agrobacterium
Please cite this article as: Syombua, E. D., Zhang, Z., Tripathi, J. N., Ntui, V. O., Kang, M., George, O. O., Edward, N. K., Wang, K., Yang, B. and Tripathi, L. (2020) A
CRISPR/Cas9-based genome-editing system for yam (Dioscorea spp.). Plant Biotechnol J., https://doi.org/10.1111/pbi.13515
ª 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. 1
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
2 Easter D. Syombua et al.
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.
ª 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–3
CRISPR/Cas-mediated genome-editing of yam 3
strain (EHA105, LBA4404), and cell density (OD600 = 0.05–2.0), Amola was 83.3% (5 mutant events out of 6 transgenic events).
supplementation of acetosyringone (200, 400 µM), and applica- These results demonstrated that the CRISPR/Cas9 could induce
tion of heat shock (Norkunas et al., 2018) to infiltrated plants site-specific disruption of the PDS gene and produced stable
were evaluated for protocol optimization. The optimal period for phenotypical changes in yam. And we expect the established
maximum infection was assessed by determining the GFP CRISPR/Cas9 system, with improved genetic transformation, will
fluorescence intensities of leaves assessed at 0, 2, 4, 6, 8, and enable function genomics and trait improvement in yam.
10 days post-infiltration (dpi). The optimized agroinfiltration
system with Agrobacterium strain EHA105 harbouring pCas9-
Acknowledgements
gRNA-PDS (OD600 = 0.75) suspended in infiltration buffer (Mur-
ashige and Skoog medium salts and vitamins, 20 g/L sucrose, This research was supported by the US NSF (IOS 1543888 to KW,
1 mg/L 6-benzylaminopurine, 0.2 µM CuSO4, pH 5.7) supple- LT, and BY), USDA NIFA (IOW04714 to KW), the State of Iowa
mented with 400 lM acetosyringone, infiltrated in the fully funds, and the ISU Crop Bioengineering Center (to KW).
expanded young leaves and heat shock treatment at 37 °C for
30 min at 2 dpi showed the highest level of transient gene Conflict of interest
expression as bleached patches and a bright GFP fluorescence at
4 dpi (Figure 1e). The observed bleached patches could be the Authors declare no conflict of interest.
results from transient knockout of the PDS gene and Agrobac-
terium infection. Authors’ contributions
To validate the efficiency of CRISPR/Cas9 for targeted muta-
genesis in stable transgenic plants, the construct pCas9-gRNA- KW, LT, and BY conceived and managed the project; LT, EDS, and
PDS was introduced into nodal explants of Amola using the JNT designed the experiments; EDS generated the edited events;
Agrobacterium-mediated transformation method developed by MK maintained yam materials and assisted in protoplast exper-
Nyaboga et al. (2014). A total of eight plants, representing 6 iments; ZZ identified yam promoters and made CRISPR constructs;
independent transgenic events, were regenerated from a total of EDS, JNT, and VON performed molecular characterization; VON
300 nodal explants over three transformation experiments. Seven and BY analysed the sequencing data; OOG and NKE participated
plants except one (#7, green) showed phenotypes of variegated in project discussion; EDS, JNT, LT, KW, and BY wrote and revised
the manuscript with contribution from other authors.
to complete albinism (Figure 1f). The variegated plants with a
mosaic pattern of albinism suggest a high level of chimerism with
mutations happening at different stages of plant regeneration. References
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ª 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–3