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Yam (Dioscorea spp.) is a multi‐species tuber crop providing food and income to millions of people worldwide, particularly in Africa (Price et al., 2016). The ‘yam belt’ in West Africa, including Nigeria, Benin, Togo, Ghana, and Côte d’Ivoire, accounts for 92% of 72.6 million tons of global yam production (FAOSTAT, 2018). Despite the economic importance, yam cultivation is plagued by several biotic and abiotic factors. Yam genetic improvement via conventional breeding has not achieved substantial progress mainly due to the dioecy nature, long breeding‐cycle, polyploidy, heterozygosity, poor seed set, and non‐synchronous flowering (Mignouna et al., 2008). A precise genome‐engineering holds the potential to overcome some of these limitations. CRISPR/Cas9 is the most popular genome‐editing system applied extensively for crop improvement, wherein yam is lagging far behind other crop species. The genetic transformation technologies and genome sequences, only recently available, made it possible to realize the potential of CRISPR‐based genome editing for basic and applied research in yam (Manoharan et al., 2016; Nyaboga et al., 2014; Tamiru et al., 2017). Here, we report, for the first time, the successful establishment of a CRISPR/Cas9‐based genome‐editing system and validation of its efficacy by targeting the phytoene desaturase gene (DrPDS) in a West African farmer‐preferred D. rotundata accession Amola. The PDS gene is involved in converting phytoene into carotenoid precursors phytofluene and ζ‐carotene (Mann et al., 1994). It is commonly used as a visual marker to validate genome editing in plants, as disruption of its function causes albinism.

We first sought to identify the promoters for expressing guide RNAs (gRNAs) in yam. Five U6 genes from D. alata were identified, and the respective promoters (~300 bp) were synthesized. To identify the best DaU6 promoters, a gRNA targeting a mutated green fluorescence protein gene (GFP + 1) was constructed under each DaU6 promoter. Individual gRNAs and Cas9 (pCas9‐DaU6::gGFP + 1) and p35S::GFP + 1 were mixed equally and introduced into the yam mesophyll protoplasts, while pUbi::GFP and p35::GFP + 1 were used as positive and negative control, respectively, using the method described previously for assessing the efficacy of wheat U6 promoters (Zhang et al., 2019). Protoplasts transfected with pUbi::GFP showed strong fluorescence 40 h post‐transfection (Figure 1a). In contrast, no fluorescence was observed in protoplasts with non‐functional GFP + 1. Some protoplasts transfected with pCas9‐DaU6::gGFP + 1 and p35S::GFP + 1 together showed GFP fluorescence, indicating the GFP + 1 was correctly edited into the functional gfp gene (Figure 1a). A comparison of the efficacy of different yam U6 promotors (DaU6.1 to DaU6.5) using the protoplast transfection assay showed variation in the number of GFP‐fluorescing protoplasts and their intensity. Promoter DaU6.5 performed best, while DaU6.2 and DaU6.3 yielded similar fluorescence scores (Figure 1b). Consequently, we selected promoters DaU6.3 and DaU6.5 to direct the gRNA expression for stable transformation of yam. The complete sequence of DrPDS was identified by Blast searching the NCBI database using Arabidopsis phytoene desaturase 3 protein (NP_193157.1) (Figure 1c). A plasmid construct, pCas9‐gRNA‐PDS, was built. This construct carries a Cas9‐gfp fusion gene driven by maize ubiquitin promoter (Zhang et al., 2019), two gRNAs targeting exon 2 of DrPDS (Figure 1c) under DaU6.3 and DaU6.5 promoters individually, and a plant selectable marker nptII gene under CaMV35S promoter (Figure 1d).

We next evaluated the efficacy of the Cas9‐gfp gene expression in yam using agroinfiltration. The agroinfiltration‐based system was established through the infiltration of young leaves of two months old potted plants with Agrobacterium harbouring the construct pCas9‐gRNA‐PDS (Figure 1d). The effect of various factors, including the age of leaves (young, unopened; young, fully expanded; and mature), infiltration buffer, Agrobacterium strain (EHA105, LBA4404), and cell density (OD₆₀₀ = 0.05–2.0), supplementation of acetosyringone (200, 400 µm), and application of heat shock (Norkunas et al., 2018) to infiltrated plants were evaluated for protocol optimization. The optimal period for maximum infection was assessed by determining the GFP fluorescence intensities of leaves assessed at 0, 2, 4, 6, 8, and 10 days post‐infiltration (dpi). The optimized agroinfiltration system with Agrobacterium strain EHA105 harbouring pCas9‐gRNA‐PDS (OD₆₀₀ = 0.75) suspended in infiltration buffer (Murashige and Skoog medium salts and vitamins, 20 g/L sucrose, 1 mg/L 6‐benzylaminopurine, 0.2 µm CuSO₄, pH 5.7) supplemented with 400 μm acetosyringone, infiltrated in the fully expanded young leaves and heat shock treatment at 37 °C for 30 min at 2 dpi showed the highest level of transient gene expression as bleached patches and a bright GFP fluorescence at 4 dpi (Figure 1e). The observed bleached patches could be the results from transient knockout of the PDS gene and Agrobacterium infection.

To validate the efficiency of CRISPR/Cas9 for targeted mutagenesis in stable transgenic plants, the construct pCas9‐gRNA‐PDS was introduced into nodal explants of Amola using the Agrobacterium‐mediated transformation method developed by Nyaboga et al. (2014). A total of eight plants, representing 6 independent transgenic events, were regenerated from a total of 300 nodal explants over three transformation experiments. Seven plants except one (#7, green) showed phenotypes of variegated 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. Some of the albino plants exhibited bushy phenotype and inadequate response to micropropagation (Figure 1f‐1, 2). However, some of the albino and variegated events produced complete plants with well‐developed roots similar to the wild‐type plants (Figure 1f‐3‐4). The expression of the transgene in these events was further confirmed by GFP fluorescence under stereomicroscope with fluorescence illuminator (Filter GFP‐B, Ex 570/40, and Em 525/50). Leaves of transgenic plants emitted a bright fluorescence (Figure 1f‐5), while wild‐type plants did not emit any fluorescence (Figure 1f‐6). All putative transgenic plants contained Cas9 as confirmed by PCR analysis.

The target region (300‐bp) of DrPDS from individual plants, with 4 leaves per plant separately sampled for DNA, was amplified by PCR using gene‐specific primers and the amplicons were subjected directly to Sanger sequencing. Plants #1 to #3 showed identical chromatographs with indels at the same locations (5‐bp deletion for gRNA1 and 1‐bp insertion for gRNA2), confirming the clonal nature of their same origin. Sequencing of other four transgenic plants (#4, 5, 6, and 8) showed various deletions (Figure 1g). All five mutant events (#1, 4, 5, 6, and 8) showed different indels proving to be independent. The indels were observed at both target sites for gRNA1 and gRNA2 in all the events within 3–4 bp upstream of the PAM sequences. Events #5, 6, and 8 carried the large deletions of 58–59 bp with sequences deleted between the cleavage sites of two gRNAs. As expected, the green plant (#7) showed no mutation at either target site. The genome‐editing efficiency in yam accession Amola was 83.3% (5 mutant events out of 6 transgenic events). These results demonstrated that the CRISPR/Cas9 could induce site‐specific disruption of the PDS gene and produced stable phenotypical changes in yam. And we expect the established CRISPR/Cas9 system, with improved genetic transformation, will enable function genomics and trait improvement in yam.

致编辑

山药（薯os）是一种多品种的块茎作物，为世界各地，尤其是非洲的数百万人提供粮食和收入（Price等，2016）。西非的``山药带''包括尼日利亚，贝宁，多哥，加纳和科特迪瓦，占全球7260万吨山药的92％（粮农组织统计数据库，2018年）。尽管在经济上很重要，但是山药的种植还是受到一些生物和非生物因素的困扰。通过传统育种进行的山药遗传改良尚未取得实质性进展，这主要是由于对雌性，繁殖周期长，多倍体，杂合性，结实性差和开花不同步（Mignouna等人，2008年）。）。精确的基因组工程具有克服这些局限性的潜力。CRISPR / Cas9是广泛用于作物改良的最流行的基因组编辑系统，其中山药远远落后于其他作物。遗传转化技术和基因组序列（仅在最近才可用）使得有可能实现基于CRISPR的基因组编辑在山药的基础和应用研究中的潜力（Manoharan等人，2016 ; Nyaboga等人，2014 ; Tamiru等人。，2017）。在这里，我们首次报告成功建立了基于CRISPR / Cas9的基因组编辑系统，并通过针对一个西非农民偏爱的D. rotundata入藏物Amola中的八氢番茄红素去饱和酶基因（DrPDS）。该PDS基因参与八氢番茄红素转化成类胡萝卜素前体的六氢番茄红素和ζ胡萝卜素（曼等人。，1994）。它通常用作视觉标记，以验证植物中的基因组编辑，因为其功能中断会导致白化病。

我们首先寻求鉴定在山药中表达引导RNA（gRNA）的启动子。D的5个U6基因。鉴定了alata，并合成了各自的启动子（〜300 bp）。为了鉴定最佳的DaU6启动子，在每个DaU6启动子下构建了一个靶向突变的绿色荧光蛋白基因（GFP + 1）的gRNA。将单个gRNA和Cas9（pCas9-DaU6 :: gGFP + 1）和p35S :: GFP + 1均匀混合并引入山药叶肉原生质体中，而pUbi :: GFP和p35 :: GFP + 1分别用作阳性和阴性分别使用先前描述的评估小麦U6启动子功效的方法进行对照（Zhang等，2019）。用pUbi :: GFP转染的原生质体在转染后40 h表现出强荧光（图1a）。相反，在非功能性GFP + 1的原生质体中未观察到荧光。一些用pCas9-DaU6 :: gGFP + 1和p35S :: GFP + 1转染的原生质体一起显示GFP荧光，表明GFP + 1被正确编辑为功能性gfp基因（图1a）。使用原生质体转染法对不同山药U6启动子（DaU6.1至DaU6.5）的功效进行比较，结果表明GFP荧光原生质体的数量及其强度存在差异。启动子DaU6.5表现最佳，而DaU6.2和DaU6.3产生相似的荧光评分（图1b）。因此，我们选择了启动子DaU6.3和DaU6.5来指导gRNA表达以稳定地转化山药。通过使用植物拟南芥八氢番茄红素去饱和酶3蛋白（NP_193157.1）进行Blast搜索NCBI数据库，鉴定了DrPDS的完整序列（图1c）。构建了质粒构建体pCas9-gRNA-PDS。该构建体带有由玉米泛素启动子驱动的Cas9-gfp融合基因（Zhang等，2019），两个gRNAs靶向的外显子2 DrPDS DaU6.3和DaU6.5启动子下（图1c）分别和植物选择标记的nptII下CaMV35S启动子基因（图1D）。

接下来，我们使用农用浸润法评估了山药中Cas9-gfp基因表达的功效。基于农业浸润的系统是通过两个月大的盆栽植物的幼叶浸入带有构建体pCas9-gRNA-PDS的农杆菌而建立的（图1d）。各种因素的影响，包括叶片的年龄（年轻，未打开；年轻，完全展开；并且成熟），浸润缓冲液，农杆菌菌株（EHA105，LBA4404）和细胞密度（OD ₆₀₀  = 0.05–2.0），乙酰丁香酮（200，400μ米），及热休克的应用（Norkunas等人。，2018）评估渗透植物的方案优化。通过确定在渗透后（dpi）的0、2、4、6、8和10天评估的叶片的GFP荧光强度来评估最大感染的最佳时期。用优化农杆菌渗入系统农杆菌菌株EHA105窝藏pCas9-gRNA-PDS（OD ₆₀₀  = 0.75）悬浮在浸润缓冲液（Murashige和Skoog培养基的盐和维生素，20g / L蔗糖，1 mg / L的6-苄氨基嘌呤，0.2μ米的CuSO ₄，pH值5.7），补充有400μ米乙酰丁香酮浸润在完全展开的幼叶中，并在2 dpi下于37°C进行30分钟的热激处理，显示出最高水平的瞬时基因表达，如漂白斑块和4 dpi处的明亮GFP荧光（图1e）。观察到的漂白斑块可能是PDS基因瞬时敲除和农杆菌感染的结果。

为了验证CRISPR / Cas9在稳定的转基因植物中定向诱变的效率，使用Nyaboga等人开发的农杆菌介导的转化方法将构建体pCas9-gRNA-PDS引入到Amola的节点外植体中。（2014年）。在三个转化实验中，从总共300个节点外植体中再生了代表6个独立转基因事件的总共8株植物。除一种植物（7号植物，绿色）外，其余7种植物表现出杂色化为完全白化病的表型（图1f）。具有白化病马赛克图案的杂色植物表明高水平的嵌合体，并且在植物再生的不同阶段发生突变。一些白化病植物表现出浓密的表型，并且对微繁殖的反应不足（图1f-1、2）。但是，一些白化病和杂色事件产生了完整的植物，其根部发达，类似于野生型植物（图1f-3-4）。在这些事件中转基因的表达进一步通过具有荧光照明器的立体显微镜在GFP荧光下得到证实（Filter GFP-B，Ex 570/40，和Em 525/50）。转基因植物的叶子发出明亮的荧光（图1f-5），而野生型植物则不发出任何荧光（图1f-6）。所有推定的转基因植物都含有通过PCR分析确认的Cas9。

DrPDS的目标区域（300-bp）使用基因特异性引物通过PCR扩增来自单个植物的单株植物，每株植物分别采集4片叶子用于DNA提取，并将扩增子直接进行Sanger测序。植物1至3显示的色谱图相同，在相同位置具有插入缺失（gRNA1缺失5 bp，gRNA2插入1 bp），证实了它们相同来源的克隆性质。其他四种转基因植物（＃4、5、6和8）的测序显示出各种缺失（图1g）。所有五个突变事件（＃1、4、5、6和8）显示出不同的indel，事实证明它们是独立的。在PAM序列上游3-4 bp内的所有事件中，在gRNA1和gRNA2的两个靶位点均观察到插入缺失。事件＃5、6和8进行了58-59 bp的大缺失，并在两个gRNA的切割位点之间删除了序列。不出所料 绿色植物（＃7）在任一目标位点均未显示突变。入山薯Amoa的基因组编辑效率为83.3％（6个转基因事件中有5个突变事件）。这些结果表明CRISPR / Cas9可以诱导特定位点的破坏。PDS基因并在山药中产生稳定的表型变化。我们希望建立的CRISPR / Cas9系统具有改进的遗传转化能力，能够使山药的功能基因组学和性状得到改善。