CRISPR/Cas9-Mediated Deletion of Large Genomic Fragments in Soybean
CRISPR/Cas9-Mediated Deletion of Large Genomic Fragments in Soybean
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DOI：
10.3390/ijms19123835
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发表时间：
2018-12-01
影响因子：
5.6
通讯作者：
Hou, Wensheng
中科院分区：
文献类型：
--
作者：
Cai, Yupeng;Chen, Li;Hou, Wensheng
目前，CRISPR/Cas9在大豆（Glycine max（L.）梅尔）主要集中于敲除靶基因，并且大多数定点诱变发生在单个切割位点并导致短缺失和/或插入。然而，使用多个指导RNA进行复杂的基因组编辑，特别是在大豆中删除大的DNA片段，尚未得到系统的探索。在这项研究中，我们采用CRISPR/Cas9技术，使用双sgRNA/Cas9设计特异性诱导大豆中GmFT 2a（Glyma 16 g26660）和GmFT 5a（Glyma 16 g 04830）中DNA片段的靶向缺失。GmFT 2a中599 - 1618 bp的靶片段的缺失频率为15.6%。我们还实现了GmFT 2a中超过4.5 kb的靶片段的12.1%的缺失频率和GmFT 5a中1069至1161 bp范围内的靶片段的15.8%的缺失频率。此外，我们证明了这些CRISPR/Cas9诱导的大片段缺失可以遗传。1618 bp缺失的无T2转基因纯合突变体表现出晚花表型。在这项研究中，我们开发了一种利用CRISPR/Cas9删除大豆大片段的有效系统;该系统将有助于未来基因功能的研究，并通过大豆染色体工程或定制遗传育种来改善农业。
At present, the application of CRISPR/Cas9 in soybean (Glycine max (L.) Merr.) has been mainly focused on knocking out target genes, and most site-directed mutagenesis has occurred at single cleavage sites and resulted in short deletions and/or insertions. However, the use of multiple guide RNAs for complex genome editing, especially the deletion of large DNA fragments in soybean, has not been systematically explored. In this study, we employed CRISPR/Cas9 technology to specifically induce targeted deletions of DNA fragments in GmFT2a (Glyma16g26660) and GmFT5a (Glyma16g04830) in soybean using a dual-sgRNA/Cas9 design. We achieved a deletion frequency of 15.6% for target fragments ranging from 599 to 1618 bp in GmFT2a. We also achieved deletion frequencies of 12.1% for target fragments exceeding 4.5 kb in GmFT2a and 15.8% for target fragments ranging from 1069 to 1161 bp in GmFT5a. In addition, we demonstrated that these CRISPR/Cas9-induced large fragment deletions can be inherited. The T2 transgene-free' homozygous ft2a mutants with a 1618 bp deletion exhibited the late-flowering phenotype. In this study, we developed an efficient system for deleting large fragments in soybean using CRISPR/Cas9; this system could benefit future research on gene function and improve agriculture via chromosome engineering or customized genetic breeding in soybean.