Targeted mutagenesis in wheat microspores using CRISPR/Cas9.
Targeted mutagenesis in wheat microspores using CRISPR/Cas9.
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DOI：
10.1038/s41598-018-24690-8
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发表时间：
2018-04-25
影响因子：
4.6
通讯作者：
Kagale S
中科院分区：
文献类型：
--
作者：
Bhowmik P;Ellison E;Polley B;Bollina V;Kulkarni M;Ghanbarnia K;Song H;Gao C;Voytas DF;Kagale S
CRISPR/Cas9基因组编辑是一项变革性的技术，将促进作物的发展，以满足未来的需求。然而，由于许多作物物种的生命周期较长，以及所需的基因类型通常需要多个世代才能实现，基因编辑的应用受到了阻碍。单细胞小孢子是一种单倍体细胞，可以发育成双单倍体植株，已被广泛用作在一代内产生纯合植株的育种工具。在本研究中，我们将CRISPR/Cas9系统与小孢子技术相结合，建立了一种优化的单倍体诱变体系，以诱导小麦基因组的遗传修饰。我们研究了一些可能影响CRISPR/CAS9试剂进入小孢子的因素，发现使用10-20 微克DNA和500 V的脉冲电压对至少75,000个细胞进行电穿孔是使用霓虹灯导入系统的最佳选择。利用多个Cas9和sgRNA构建，我们提出了在一个外源DsRed基因和两个内源小麦基因(包括TaLox2和TaUbiL1)中无缝引入靶向修饰的证据。本研究论证了小孢子技术与基于CRISPR/Cas9的基因编辑技术相结合用于植物性状发现和改良的价值和可行性。
CRISPR/Cas9 genome editing is a transformative technology that will facilitate the development of crops to meet future demands. However, application of gene editing is hindered by the long life cycle of many crop species and because desired genotypes generally require multiple generations to achieve. Single-celled microspores are haploid cells that can develop into double haploid plants and have been widely used as a breeding tool to generate homozygous plants within a generation. In this study, we combined the CRISPR/Cas9 system with microspore technology and developed an optimized haploid mutagenesis system to induce genetic modifications in the wheat genome. We investigated a number of factors that may affect the delivery of CRISPR/Cas9 reagents into microspores and found that electroporation of a minimum of 75,000 cells using 10–20 µg DNA and a pulsing voltage of 500 V is optimal for microspore transfection using the Neon transfection system. Using multiple Cas9 and sgRNA constructs, we present evidence for the seamless introduction of targeted modifications in an exogenous DsRed gene and two endogenous wheat genes, including TaLox2 and TaUbiL1. This study demonstrates the value and feasibility of combining microspore technology and CRISPR/Cas9-based gene editing for trait discovery and improvement in plants.
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影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
3
作者：
Eudes, F;Acharya, S;Cheng, KJ
通讯作者：
Cheng, KJ
影响因子：
2.3
作者：
Sadastivaiah, RS;Perkovic, SM;Postman, B
通讯作者：
Postman, B
DOI：
10.1111/tpj.13446
发表时间：
2017-03
期刊：
The Plant journal : for cell and molecular biology
影响因子：
--
作者：
Gil-Humanes J;Wang Y;Liang Z;Shan Q;Ozuna CV;Sánchez-León S;Baltes NJ;Starker C;Barro F;Gao C;Voytas DF
通讯作者：
Voytas DF