Target base editing in soybean using a modified CRISPR/Cas9 system.
Target base editing in soybean using a modified CRISPR/Cas9 system.
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使用改良的 CRISPR/Cas9 系统对大豆进行靶向碱基编辑。
DOI：
10.1111/pbi.13386
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发表时间：
2020-10
影响因子：
13.8
通讯作者：
Hou W
中科院分区：
文献类型：
--
作者：
Cai Y;Chen L;Zhang Y;Yuan S;Su Q;Sun S;Wu C;Yao W;Han T;Hou W
近年来，CRISPR（成簇的规则间隔短回文重复序列）/Cas9（CRISPR相关9）系统由于其简单、高效、成本效益和多功能性的优点，已经彻底改变了功能基因组研究和作物改良（Chen et al. 2019年）。在植物中已经有大量的报道显示，通过非同源末端连接（NHEJ）在预期靶位点处的少量碱基插入/缺失（indel）通常用于通过移码突变破坏基因功能（Chen等人，2019年）。最近，已经从CRISPR/Cas9系统的修饰开发了一种称为“碱基编辑”的新的和强大的策略，其使得能够通过RNA编程的方式将单个碱基取代成另一个碱基，而不需要DNA双链断裂或供体模板（Komor et al. 2016年）。Cas9可以通过使其两个内切核酸酶结构域中的任一个失活而被修饰以充当切口酶（Cas9 n）。据报道，带切口的Cas9（D10 A突变）与胞苷脱氨酶和尿嘧啶糖基化酶抑制剂（UGI）的融合通常允许在靶序列的小窗口（从位置4至8，将远离PAM（前间区序列相邻基序）的末端计数为位置1）内C至T（或G至A）取代（Komor等人，2016年; Li等人，2017年）的报告。许多重要的农业性状与单核苷酸多态性（SNP）变异相关。利用功能性SNP也是改良作物农艺性状的重要手段。因此，在与多种重要农艺性状相关的特定位点产生点突变在分子育种中具有重要价值（Mishra et al.，2020年）。例如，在水稻中使用碱基编辑，以1.4%-11.5%的频率实现了C到T的替换，并且还以1.6%-3.9%的频率检测到C到G的变化。此外，具有C至T取代的SLR 1突变体（S97 L）显示出明显的半矮秆表型（Lu和Zhu，2017）。此外，还报道了两种重要作物玉米和小麦中的碱基编辑事件（Zong等人，2017年）。然而，在大豆（Glycine max（L.）Merr.），大豆是一种重要的豆类作物，具有巨大的经济价值，为粮食生产和动物饲料提供丰富的蛋白质和油脂。在这项研究中，我们开发了一种CRISPR/Cas9介导的碱基编辑工具，以特异性地诱导大豆中的单碱基替换。我们将Cas9 n（D10 A）切口酶、大鼠胞嘧啶脱氨酶（APOBEC 1）和尿嘧啶糖基化酶抑制剂（UGI）组合作为碱基编辑器（BE），然后将这些元件克隆到pTF 101中。1个载体以产生pTF 101。1-BE。BE由2X CaMV 35 S启动子驱动。sgRNA表达盒的表达由pUC 57载体内的拟南芥U6启动子驱动，从而产生pUC 57-sgRNA。一旦选择了所需的靶序列，仅需要克隆编码sgRNA的DNA序列。从pUC 57-sgRNA切割含有所需sgRNA序列的表达盒并插入pTF 101中。1-BE以产生pTF 101。1-sgRNA-BE载体，其将用于尝试单碱基取代。已经在大豆中鉴定了10个FLOWERNLOCUS T（FT）同源物（Kong等人，2010年）。为了验证大豆中碱基编辑载体的可行性和有效性，我们选择了GmFT 2a（Glyma. 16 G150700）和GmFT 4（Glyma. 08 G363100）作为靶基因。靶序列分别位于GmFT 2a的第一外显子和GmFT 4的第四外显子（图1a）。相应的pTF 101。将1-sgRNA-BE载体转化到...
In recent years, the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated 9) system has revolutionized functional genomic research and crop improvement, owing to its advantages of simplicity, efficiency, cost-effectiveness and versatility (Chen et al., 2019). There have been abundant reports in plants showing that a small amount of base insertions/deletions (indels) at the intended target site through non-homologous end joining (NHEJ) are typically used to disrupt gene function by frameshift mutations (Chen et al., 2019). Recently, a new and powerful strategy called ‘base editing’has been developed from modifications of the CRISPR/Cas9 system, which enables single base substitution into another through an RNA-programmed manner, without requiring DNA double-strand breaks or a donor template (Komor et al., 2016). Cas9 can be modified to serve as a nickase enzyme (Cas9n) by inactivating either of its two endonuclease domains. It has been reported that the fusion of a nicked Cas9 (D10A mutation) with a cytidine deaminase enzyme and an uracil glycosylase inhibitor (UGI) typically allows C to T (or G to A) substitution within a small window (from positions 4 to 8, counting the end distal to the PAM (protospacer adjacent motif) as position 1) of the target sequence (Komor et al., 2016; Li et al., 2017). Many agriculturally important traits are associated with single nucleotide polymorphism (SNP) variation. Utilization of functional SNPs is also an important means to improve agronomic characters of crops. Therefore, generation of point mutations at specific sites associated with diverse important agronomic traits is of great value in molecular breeding (Mishra et al., 2020). For instance, using base editing in rice, C to T substitution was achieved at a frequency of 1.4%–11.5%, and C to G change was also detected at a frequency of 1.6%–3.9%. Moreover, the SLR1 mutants with C to T substitution (S97L) display obvious semi-dwarf phenotypes (Lu and Zhu, 2017). In addition, base editing events in two important crops, maize and wheat, have also been reported (Zong et al., 2017). However, it has not been systematically explored in soybean (Glycine max (L.) Merr.), an important legume crop with great economic value that provides abundant protein and oil for food production and animal feed.In this study, we developed a CRISPR/Cas9-mediated base editing tool to specifically induce single base substitution in soybean. We combined the Cas9n (D10A) nickase, rat cytosine deaminase (APOBEC1) and uracil glycosylase inhibitor (UGI) as the base editor (BE), and then cloned these elements into the pTF101. 1 vector to generate pTF101. 1-BE. BE was driven by a 2X CaMV 35S promoter. Expression of the sgRNA expression cassettes was driven by the Arabidopsis U6 promoter within the pUC57 vector, thus generating the pUC57-sgRNA. Once a desired target sequence is selected, only the DNA sequence encoding the sgRNA needs to be cloned. The expression cassettes containing desired sgRNA sequences were cut from pUC57-sgRNA and inserted into pTF101. 1-BE to generate the pTF101. 1-sgRNA-BE vector, which would be used to attempt single base substitution. Ten FLOWERING LOCUS T (FT) homologs have been identified in soybean (Kong et al., 2010). To verify the base editing vector in soybean for its feasibility and efficacy, we selected GmFT2a (Glyma. 16G150700) and GmFT4 (Glyma. 08G363100) as target genes. The target sequences were located in the first exon of GmFT2a and fourth exon of GmFT4, respectively (Figure 1a). The corresponding pTF101. 1-sgRNA-BE vectors were transformed into …
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作者：
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