Efficient identification of gene knockout mutant mediated by CRISPR/Cas9 by CELⅠ crude extracts

Journal Article / 2017

Luo, Wanbing; Lin, Qiupeng; Li, Xiaoxia; Zhou, Zejiao; Yang, Huiyong; Du, Rongyu; Li, Hongqing

CRISPR/Cas9, emerged as an efficient and powerful gene editing technology, has become the mainstream genome editing technology. Constructing mutants using CRISPR/Cas9 system is of great significance to the functional study and breeding application of useful genes. As the basis of the technology, a method for identification of mutation with efficiency and lower cost is needed. In this report, we studied the factors influencing mutation detected by CEL Ⅰ crude extracts, such as the amount of protein, enzyme incubation time, PCR buffers. Under the optimized conditions, we can integrate the mutation detection steps into one-tube reaction. We used this system to examine the mutation types and frequency of rice stn1 mediated by CRISPR/Cas9. We also used this method to identify different mutation types including homozygous, heterozygous and bi-allelic mutations. The accuracy of this method reached 100% verified by sequencing. Altogether, our results showed that using CELⅠ crude extracts was an efficient and low cost method for identification of CRISPR/Cas9 mediated mutation. CRISPR/Cas9 是新兴的基因组编辑技术，该技术操作简单、效率高，已成为目前最主流的基因组编辑技术。利用该技术进行突变体创制，对基因功能研究和育种应用具有重要的意义，而快速、高效、低成本的基因编辑个体鉴定是其中的重要环节。本研究对影响CELⅠ粗提物鉴定CRISPR/Cas9 介导的水稻基因编辑个体的条件，包括蛋白用量及作用时间、PCR 反应缓冲液等条件进行了探索，并将整个检测体系集成于一管操作。同时，采用CELⅠ粗提物检测了CRISPR/Cas9 介导水稻stn1 突变的T₀ 代植株及后代，对杂合突变、纯合突变及双等位突变的鉴定策略进行了探讨；该方法检测正确率经测序验证达100%。上述结果表明，采用CELⅠ粗提物检测突变体与已有方法比较具有廉价、快速和高效的特点。.