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Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice.
Communications Biology ( IF 5.2 ) Pub Date : 2020-01-23 , DOI: 10.1038/s42003-020-0768-9 Zahir Ali 1 , Ashwag Shami 1, 2 , Khalid Sedeek 1 , Radwa Kamel 1 , Abdulrahman Alhabsi 1 , Muhammad Tehseen 3 , Norhan Hassan 1 , Haroon Butt 1 , Ahad Kababji 1 , Samir M Hamdan 3 , Magdy M Mahfouz 1
Communications Biology ( IF 5.2 ) Pub Date : 2020-01-23 , DOI: 10.1038/s42003-020-0768-9 Zahir Ali 1 , Ashwag Shami 1, 2 , Khalid Sedeek 1 , Radwa Kamel 1 , Abdulrahman Alhabsi 1 , Muhammad Tehseen 3 , Norhan Hassan 1 , Haroon Butt 1 , Ahad Kababji 1 , Samir M Hamdan 3 , Magdy M Mahfouz 1
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Precise genome editing by systems such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) requires high-efficiency homology-directed repair (HDR). Different technologies have been developed to improve HDR but with limited success. Here, we generated a fusion between the Cas9 endonuclease and the Agrobacterium VirD2 relaxase (Cas9-VirD2). This chimeric protein combines the functions of Cas9, which produces targeted and specific DNA double-strand breaks (DSBs), and the VirD2 relaxase, which brings the repair template in close proximity to the DSBs, to facilitate HDR. We successfully employed our Cas9-VirD2 system for precise ACETOLACTATE SYNTHASE (OsALS) allele modification to generate herbicide-resistant rice (Oryza sativa) plants, CAROTENOID CLEAVAGE DIOXYGENASE-7 (OsCCD7) to engineer plant architecture, and generate in-frame fusions with the HA epitope at HISTONE DEACETYLASE (OsHDT) locus. The Cas9-VirD2 system expands our ability to improve agriculturally important traits in crops and opens new possibilities for precision genome engineering across diverse eukaryotic species.
中文翻译：
Cas9核酸内切酶和VirD2松弛酶的融合促进了针对水稻基因组工程的同源性修复。
通过诸如规则间隔的短回文重复序列（CRISPR）/ CRISPR相关蛋白9（Cas9）之类的系统进行精确的基因组编辑需要高效的同源性定向修复（HDR）。已经开发了多种技术来改善HDR，但效果有限。在这里，我们在Cas9核酸内切酶和农杆菌VirD2松弛酶（Cas9-VirD2）之间产生了融合体。该嵌合蛋白结合了Cas9的功能（可产生有针对性的DNA特异双链断裂（DSB））和VirD2松弛酶（可将修复模板与DSB紧密结合）来促进HDR。我们成功地将Cas9-VirD2系统用于精确的乙酰乳酸合酶（OsALS）等位基因修饰，以产生抗除草剂的水稻（Oryza sativa）植物，类胡萝卜素切割DIOXYGENASE-7（OsCCD7）用于构建植物结构，并在HISTONE DEACETYLASE（OsHDT）基因座上与HA表位产生框内融合。Cas9-VirD2系统扩展了我们改善农作物重要农艺性状的能力，并为跨各种真核生物物种进行精确基因组工程打开了新的可能性。
更新日期：2020-01-23
中文翻译：
Cas9核酸内切酶和VirD2松弛酶的融合促进了针对水稻基因组工程的同源性修复。
通过诸如规则间隔的短回文重复序列（CRISPR）/ CRISPR相关蛋白9（Cas9）之类的系统进行精确的基因组编辑需要高效的同源性定向修复（HDR）。已经开发了多种技术来改善HDR，但效果有限。在这里，我们在Cas9核酸内切酶和农杆菌VirD2松弛酶（Cas9-VirD2）之间产生了融合体。该嵌合蛋白结合了Cas9的功能（可产生有针对性的DNA特异双链断裂（DSB））和VirD2松弛酶（可将修复模板与DSB紧密结合）来促进HDR。我们成功地将Cas9-VirD2系统用于精确的乙酰乳酸合酶（OsALS）等位基因修饰，以产生抗除草剂的水稻（Oryza sativa）植物，类胡萝卜素切割DIOXYGENASE-7（OsCCD7）用于构建植物结构，并在HISTONE DEACETYLASE（OsHDT）基因座上与HA表位产生框内融合。Cas9-VirD2系统扩展了我们改善农作物重要农艺性状的能力，并为跨各种真核生物物种进行精确基因组工程打开了新的可能性。
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