PUBLICATION
A simple and efficient method for CRISPR/Cas9-induced mutant screening
- Authors
- Hua, Y., Wang, C., Huang, J., Wang, K.
- ID
- ZDB-PUB-170419-8
- Date
- 2017
- Source
- Journal of genetics and genomics = Yi chuan xue bao 44(4): 207-213 (Journal)
- Registered Authors
- Huang, Jian
- Keywords
- ACT-PCR, CRISPR/Cas9, Genome editing, Mutant screening
- MeSH Terms
-
- Animals
- CRISPR-Cas Systems/genetics*
- DNA Mutational Analysis/methods*
- Oryza/genetics
- Polymerase Chain Reaction
- Zebrafish/genetics
- PubMed
- 28416245 Full text @ J. Genet. Genomics
Citation
Hua, Y., Wang, C., Huang, J., Wang, K. (2017) A simple and efficient method for CRISPR/Cas9-induced mutant screening. Journal of genetics and genomics = Yi chuan xue bao. 44(4):207-213.
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping