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 cha...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 timeand 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.展开更多
基金supported by the National Natural Science Foundation of China (Nos. 31271681 and 3140101312)the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences
文摘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 timeand 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.
