Efficient identification of gene knockout mutant mediated by CRISPR/Cas9 by CELⅠ crude extracts / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 775-784, 2017.
Article
in Chinese
| WPRIM
| ID: wpr-242230
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Type of study:
Diagnostic study
/
Prognostic study
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2017
Type:
Article
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