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.
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.
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DOI：
10.3390/ijms23094889
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发表时间：
2022-04-28
影响因子：
5.6
通讯作者：
中科院分区：
文献类型：
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Golden gate/模块化克隆通过利用IIS型限制性内切酶的独特功能，促进更快和更有效的克隆。然而，已知DNA片段的靶向插入必须不包括内部IIS型限制性识别位点。在通过使用金门（GG）克隆来克隆CRISPR构建体的情况下，这缩小了向导RNA（gRNA）挑选的范围，因为选择用于成功基因组编辑的良好gRNA需要履行一些义务，并且如果仅因为其具有内部IIS型限制性识别位点而不能挑选良好gRNA候选物，则这是不希望的。在这篇文章中，我们已经证明了gRNA中IIS型限制性识别位点的存在不会影响克隆和随后的基因组编辑。在GG反应的每个步骤后，通过菌落颜色和限制性消化验证gRNA的正确插入，并通过测序进一步确认。最后，将含有Cas 12 a核酸酶和四种gRNA的最终载体用于农杆菌介导的柑橘细胞转化。对gRNA-2侧翼的PCR扩增子的测序显示转基因植物中的取代（C至T）突变。从这项研究中获得的知识可以拓宽GG克隆的范围，特别是用于GG介导的克隆到CRISPR载体中的gRNA选择的范围。
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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DOI：
10.1007/978-1-0716-0712-1_19
发表时间：
2020-01-01
期刊：
RNA TAGGING
影响因子：
--
作者：
Schindele, Patrick;Wolter, Felix;Puchta, Holger
通讯作者：
Puchta, Holger
影响因子：
13.8
作者：
Jia, Hongge;Orbovic, Vladimir;Wang, Nian
通讯作者：
Wang, Nian
影响因子：
6.2
作者：
Zhang, Fei;LeBlanc, Chantal;Jacob, Yannick
通讯作者：
Jacob, Yannick
影响因子：
14.9
作者：
Lin D;O'Callaghan CA
通讯作者：
O'Callaghan CA
影响因子：
14.9
作者：
Concordet JP;Haeussler M
通讯作者：
Haeussler M