A simple and efficient method for CRISPR/Cas9-induced mutant screening

Yufeng Hua; Chun Wang; Jian Huang; Kejian Wang

doi:10.1016/j.jgg.2017.03.005

Volume 44 Issue 4

Apr. 2017

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Article Navigation > Journal of Genetics and Genomics > 2017 > 44(4): 207-213

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A simple and efficient method for CRISPR/Cas9-induced mutant screening

doi: 10.1016/j.jgg.2017.03.005

Yufeng Hua^{a, #},
Chun Wang^{a, #},
Jian Huang^{b, #},
Kejian Wang^a

a
State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310006, China
b
School of Biology & Basic Medical Sciences, Soochow University, Suzhou 215123, China

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Corresponding author: E-mail address: wangkejian@caas.cn (Kejian Wang)
Received Date: 2017-01-09
Accepted Date: 2017-03-07
Rev Recd Date: 2017-02-17

Available Online: 2017-04-04

Publish Date: 2017-04-20

Abstract

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system provides a technological breakthrough in mutant generation. Several methods such as the polymerase chain reaction (PCR)/restriction enzyme (RE) assay, T7 endonuclease I (T7EI) assay, Surveyor nuclease assay, PAGE-based genotyping assay, and high-resolution melting (HRM) analysis-based assay have been developed for screening CRISPR/Cas9-induced mutants. However, these methods are time- and labour-intensive and may also be sequence-limited or require very expensive equipment. Here, we described a cost-effective and sensitive screening technique based on conventional PCR, annealing at critical temperature PCR (ACT-PCR), for identifying mutants. ACT-PCR requires only a single PCR step followed by agarose gel electrophoresis. We demonstrated that ACT-PCR accurately distinguished CRISPR/Cas9-induced mutants from wild type in both rice and zebrafish. Moreover, the method can be adapted for accurately determining mutation frequency in cultured cells. The simplicity of ACT-PCR makes it particularly suitable for rapid, large-scale screening of CRISPR/Cas9-induced mutants in both plants and animals.
- ACT-PCR,
- CRISPR/Cas9,
- Genome editing,
- Mutant screening

These authors contributed equally to this work.

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References(17)

References

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