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Versatile in vitro assay to recognize Cas9‐induced mutations
Plant Direct ( IF 3 ) Pub Date : 2020-09-28 , DOI: 10.1002/pld3.269
Heinrich Bente 1 , Ortrun Mittelsten Scheid 1 , Mattia Donà 1
Affiliation  

The discovery of CRISPR/Cas9 has revolutionized molecular biology, and its impact on plant biotechnology and plant breeding cannot be over‐estimated. In many plant species, its application for mutagenesis is now a routine procedure––if suitable target sites, sufficient expression of the Cas9 protein, and functioning sgRNAs are combined. sgRNAs differ in their efficiency, depending on parameters that are only poorly understood. Several software tools and experience from growing databases are supporting the design of sgRNAs, but some seemingly perfect sgRNAs turn out to be inefficient or fail entirely, and most data bases stem from work with mammalian cells. Different in vitro assays testing sgRNAs in reconstituted Cas9 complexes are available and useful to reduce the risk of failure, especially in plants when CRISPR/Cas9 application requires modifications within the germ line and laborious transformation protocols. Low sgRNA efficiency and long generation times in plants can also contribute to the workload and costs of screening for the wanted genome edits. Here, we present a protocol in which a simple, initial in vitro test for suitable sgRNAs is modified to accelerate genotyping of Cas9‐induced mutations. We demonstrate applicability of our protocol for mutagenesis and mutation screen for specific genes in Arabidopsis, but the principle should be universally suitable to provide a simple, low‐cost, and rapid method to identify edited genes also in other plants and other organisms.

中文翻译:

多功能体外测定法,可识别Cas9诱导的突变

CRISPR / Cas9的发现彻底改变了分子生物学,其对植物生物技术和植物育种的影响不可高估。在许多植物物种中,将其应用于诱变已成为一种常规程序-如果适当的靶位点,足够的Cas9蛋白表达和功能性sgRNA结合在一起。sgRNA的效率各不相同,具体取决于尚不清楚的参数。不断增长的数据库中有几种软件工具和丰富的经验支持sgRNA的设计,但是一些看似完美的sgRNA却效率低下或完全失效,而且大多数数据库都来自哺乳动物细胞的工作。提供了多种体外试验来检测重组Cas9复合物中的sgRNA,这些方法有助于降低失败的风险,特别是在CRISPR / Cas9应用需要在种系内进行修饰且费力的转化方案的植物中。植物中较低的sgRNA效率和较长的生成时间也会增加筛选所需基因组编辑的工作量和成本。在这里,我们提出了一种协议,其中修改了适用于sgRNA的简单的初始体外测试,以加速Cas9诱导的突变的基因分型。我们证明了本协议适用于拟南芥中特定基因的诱变和突变筛选的适用性,但该原理应普遍适用于提供一种简单,低成本,快速的方法来鉴定其他植物和其他生物中的编辑基因。植物中较低的sgRNA效率和较长的生成时间也会增加筛选所需基因组编辑的工作量和成本。在这里,我们提出了一种协议,其中修改了适用于sgRNA的简单的初始体外测试,以加速Cas9诱导的突变的基因分型。我们证明了本协议适用于拟南芥中特定基因的诱变和突变筛选的适用性,但该原理应普遍适用于提供一种简单,低成本,快速的方法来鉴定其他植物和其他生物中的编辑基因。植物中较低的sgRNA效率和较长的生成时间也会增加筛选所需基因组编辑的工作量和成本。在这里,我们提出了一种协议,其中修改了适用于sgRNA的简单的初始体外测试,以加速Cas9诱导的突变的基因分型。我们证明了本协议适用于拟南芥中特定基因的诱变和突变筛选的适用性,但该原理应普遍适用于提供一种简单,低成本,快速的方法来鉴定其他植物和其他生物中的编辑基因。
更新日期:2020-09-29
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