Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 &ITin vitro&IT transcripts or ribonucleoproteins
Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 &ITin vitro&IT transcripts or ribonucleoproteins
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使用 CRISPR/Cas9 基因枪技术对面包小麦进行基因组编辑
DOI：
10.1038/nprot.2017.145
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发表时间：
2018-03-01
期刊：
影响因子：
14.8
通讯作者：
Gao, Caixia
中科院分区：
文献类型：
--
作者：
Liang, Zhen;Chen, Kunling;Gao, Caixia
近年来，CRISPR/Cas9已成为改善作物性状的有力工具。传统的植物基因组编辑主要依赖于通过农杆菌或粒子轰击递送的携带质粒的盒。在这里，我们描述了通过粒子轰击递送CRISPR/Cas9的体外转录物（IVT）或核糖核蛋白复合物（RNP）来对面包小麦进行无DNA编辑。该方案作为我们先前发表的使用通过粒子轰击递送的CRISPR/Cas9质粒在面包小麦中进行基因组编辑的方案的扩展。我们描述的方法不仅消除了CRISPR/Cas9随机整合到基因组DNA中，而且还减少了脱靶效应。在这篇协议扩展文章中，我们提出了详细的IVT和RNP的制备方案;通过PCR/限制性酶（RE）和下一代测序进行验证;通过生物射弹进行交付;通过合并方法和桑格测序回收突变体并鉴定突变体。要使用这些协议，研究人员应该有基本的技能和经验，在分子生物学和biolistic转化。通过使用这些方案，可以在9-11周内生成和鉴定不使用任何外源DNA编辑的植物。
In recent years, CRISPR/Cas9 has emerged as a powerful tool for improving crop traits. Conventional plant genome editing mainly relies on ptasmid-carrying cassettes delivered by Agrobacterium or particle bombardment. Here, we describe DNA-free editing of bread wheat by delivering in vitro transcripts (IVTs) or ribonucleoprotein complexes (RNPs) of CRISPR/Cas9 by particle bombardment. This protocol serves as an extension of our previously published protocol on genome editing in bread wheat using CRISPR/Cas9 plasmids delivered by particle bombardment. The methods we describe not only eliminate random integration of CRISPR/Cas9 into genomic DNA, but also reduce off-target effects. In this protocol extension article, we present detailed protocols for preparation of IVTs and RNPs; validation by PCR/restriction enzyme (RE) and next-generation sequencing; delivery by biolistics; and recovery of mutants and identification of mutants by pooling methods and Sanger sequencing. To use these protocols, researchers should have basic skills and experience in molecular biology and biolistic transformation. By using these protocols, plants edited without the use of any foreign DNA can be generated and identified within 9-11 weeks.