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Having a Same Type IIS Enzyme's Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis

文献类型： 外文期刊

作者： Moniruzzaman, M. 1 ; Zhong, Yun 1 ; Huang, Zhifeng 1 ; Zhong, Guangyan 1 ;

作者机构： 1.Guangdong Acad Agr Sci, Inst Fruit Tree Res, Guangzhou 510640, Peoples R China

2.Florida A&M Univ, Ctr Viticulture & Small Fruit Res, Tallahassee, FL 32308 USA

3.Minist Agr & Rural Affairs, Key Lab South Subtrop Fruit Biol & Genet Resource, Guangdong Prov Key Lab Trop & Subtrop Fruit Tree, Guangzhou 510640, Peoples R China

关键词： gRNA designing; modular cloning; restriction digestion; genome editing; base editing; plant transformation

期刊名称：INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES （ 影响因子：6.208； 五年影响因子：6.628 ）

ISSN：

年卷期： 2022 年 23 卷 9 期

页码：

收录情况： SCI

摘要： Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a Cas12a nuclease and four gRNAs was used for Agrobacterium-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors.

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