Using Staphylococcus aureus Cas9 to Expand the Scope of Potential Gene Targets for Genome Editing in Soybean.
Using Staphylococcus aureus Cas9 to Expand the Scope of Potential Gene Targets for Genome Editing in Soybean.
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DOI：
10.3390/ijms232112789
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发表时间：
2022-10-24
影响因子：
5.6
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中科院分区：
文献类型：
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CRISPR（clustered regularly interspaced short palindromic repeat）/Cas（CRISPR associated）是一种革命性的基因组编辑技术，已用于在包括大豆（Glycine max）在内的许多植物物种中实现位点特异性基因敲除、大片段缺失或碱基编辑。目前，化脓性链球菌Cas9（SpCas 9）被广泛用于植物中，尽管有一些报道描述了CRISPR/Cpf 1在大豆中的应用。因此，目前大豆PAM（protospacer adjacent motif）序列的选择范围仅限于5′-NGG-3′（SpCas 9）或5′-TTTN-3′（Cpf 1），这反过来又限制了可突变基因的数量。来自金黄色葡萄球菌的另一种Cas9酶（SaCas 9）识别PAM序列5′-NNGRRT-3′（其中R代表A或G），其可以提供更广泛的潜在靶序列。在这项研究中，我们开发了CRISPR/SaCas 9系统，并使用该工具在大豆毛状根中GmFT 2a（Glyma.16G150700）和GmFT 5a（Glyma.16G044100）基因的五个靶位点特异性诱导靶向突变。结果表明，该工具能够识别大豆PAM序列5′-AAGGGT-3′、5′-GGGGAT-3′、5′-TTGAAT-3′和5′-TAGGGT-3′，突变率在34.5%~ 73.3%之间。我们的研究结果表明，我们已经建立了一个高效的CRISPR/SaCas 9工具，它与SpCas 9一样适用于大豆基因组编辑，它将有助于扩大基因组编辑的靶序列范围。
The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) is a revolutionary genome editing technology that has been used to achieve site-specific gene knock-out, large fragment deletion, or base editing in many plant species including soybean (Glycine max). The Streptococcus pyogenes Cas9 (SpCas9) is widely used in plants at present, although there are some reports describing the application of CRISPR/Cpf1 in soybean. Therefore, the selection range of PAM (protospacer adjacent motif) sequences for soybean is currently limited to 5′-NGG-3′ (SpCas9) or 5′-TTTN-3′ (Cpf1), which in turn limits the number of genes that can be mutated. Another Cas9 enzyme from Staphylococcus aureus (SaCas9) recognizes the PAM sequence 5′-NNGRRT-3′ (where R represents A or G), which can provide a wider range of potential target sequences. In this study, we developed a CRISPR/SaCas9 system and used this tool to specifically induce targeted mutations at five target sites in the GmFT2a (Glyma.16G150700) and GmFT5a (Glyma.16G044100) genes in soybean hairy roots. We demonstrated that this tool can recognize the PAM sequences 5′-AAGGGT-3′, 5′-GGGGAT-3′, 5′-TTGAAT-3′, and 5′-TAGGGT-3′ in soybean, and it achieved mutation rates ranging from 34.5% to 73.3%. Our results show that we have established a highly efficient CRISPR/SaCas9 tool that is as suitable as SpCas9 for genome editing in soybean, and it will be useful for expanding the range of target sequences for genome editing.
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