Luo Wanbing, Lin Qiupeng, Li Xiaoxia, Zhou Zejiao, Yang Huiyong, Du Rongyu, Li Hongqing
Guangdong Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, Guangdong, China.
Sheng Wu Gong Cheng Xue Bao. 2017 May 25;33(5):775-784. doi: 10.13345/j.cjb.160388.
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.
