Nature Protocols∣中科院遺傳發育所高彩霞研究組利用CRISPR/Cas9 IVT或RNP實現小麥基因組編輯

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文章詳細闡述了通過基因槍將CRISPR/Cas9 IVT和RNP導入小麥未成熟幼胚實現基因組定點修飾的DNA-free基因組編輯體系。介紹了整個流程包括CRISPR/Cas9 IVT和RNP的製備、編輯活性體外及原生質體驗證、基因槍幼胚轉化法、突變體群體的快速篩選鑒定等具體方法和實驗細節。該方法只需要9-11周即可在T0代就獲得不含外源DNA的靶向修飾小麥突變體。由於IVT和RNP是以瞬時表達的方式發揮作用，成功避免外源DNA整合到宿主基因組中而且大大降低脫靶效應。該DNA-free基因組編輯體系的建立將有助於進一步完善作物基因組編輯技術，推進基因組編輯育種產業化進程。

該研究成果於2018年2月1日在線發表於Nature Protocols雜誌上（DOI:10.1038/nprot.2017.145）。高彩霞研究組博士生梁振和副研究員陳坤玲為該論文的並列第一作者。相關研究得到科技部、農業部、中科院以及國家自然基金委的資助。

Abstract：

In 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.

Figure 1 | Overview of CRISPR/Cas9 IVT- or RNP-mediated genome editing in wheat.

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