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

Wanbing Luo; Qiupeng Lin; Xiaoxia LI; Zejiao Zhou; Huiyong Yang; Rongyu DU; Hongqing LI

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

Author: Wanbing LUO ¹ ; Qiupeng LIN ¹ ; Xiaoxia LI ¹ ; Zejiao ZHOU ¹ ; Huiyong YANG ¹ ; Rongyu DU ¹ ; Hongqing LI ¹
Author Information

1. Guangdong Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, Guangdong, China.
Publication Type:Journal Article
Keywords: CELⅠ crude extracts; CRISPR/Cas9; identification; mutant; stn1
From: Chinese Journal of Biotechnology 2017;33(5):775-784
CountryChina
Language:Chinese
Abstract: 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.