This protocol is an extension to: Nat. Protoc. 9, 2395-2410 (2014); doi:10.1038/nprot.2014.157; published online 18 September 2014In recent years, CRISPR/Cas9 has emerged as a powerful tool for improving crop traits. Conventional plant genome editing mainly relies on plasmid-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.

中文翻译：

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使用CRISPR / Cas9体外转录本或核糖核蛋白的射弹传递对面包小麦进行基因组编辑。

该协议是对以下内容的扩展：Nat。协议。9，2395-2410（2014）; doi：10.1038 / nprot.2014.157; 于2014年9月18日在线发表近年来，CRISPR / Cas9已成为改善作物性状的强大工具。常规的植物基因组编辑主要依靠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传递CRISPR / Cas9的体外转录本（IVTs）或核糖核蛋白复合物（RNPs），从而实现了无面包小麦的无DNA编辑。该协议是我们先前发布的关于使用粒子轰击递送的CRISPR / Cas9质粒在面包小麦中进行基因组编辑的协议的扩展。我们描述的方法不仅消除了CRISPR / Cas9向基因组DNA的随机整合，而且减少了脱靶效应。在此协议扩展文章中，我们提出了准备IVT和RNP的详细协议；通过PCR /限制酶（RE）和下一代测序进行验证；通过生物弹药运送；融合方法和桑格测序技术鉴定和鉴定突变体。要使用这些协议，研究人员应该具有分子生物学和生物枪法转化的基本技能和经验。通过使用这些协议，可以在9-11周内生成并鉴定未经任何外源DNA编辑的植物。研究人员应具有分子生物学和生物射弹转化的基本技能和经验。通过使用这些协议，可以在9-11周内生成并鉴定未经任何外源DNA编辑的植物。研究人员应具有分子生物学和生物射弹转化的基本技能和经验。通过使用这些协议，可以在9-11周内生成并鉴定未经任何外源DNA编辑的植物。