Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system provides a technological breakthrough in targeted mutagenesis. However, a significant amount of time and cost is required to screen for the CRISPR/Cas9-induced mutants from a typically large number of initial samples. Here, we describe a cost-effective and sensitive screening technique based on conventional polymerase chain reaction (PCR), termed “annealing at critical temperature PCR” (ACT-PCR), for identifying mutants. ACT-PCR requires only a single PCR step followed by agarose gel electrophoresis. The simplicity of ACT-PCR makes it particularly suitable for rapid, large-scale screening of CRISPR/Cas9-induced mutants.
Key words
- ACT-PCR
- CRISPR/Cas9
- Genome editing
- Mutant screening
- Rapid
- Large-scale
- Cost-effective
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References
Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821
Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826
Hua Y, Wang C, Huang J et al (2017) A simple and efficient method for CRISPR/Cas9-induced mutant screening. J Genet Genomics 44:207–213
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Nos. 31271681 and 31401363) and the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences.
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Wang, C., Wang, K. (2019). Rapid Screening of CRISPR/Cas9-Induced Mutants Using the ACT-PCR Method. In: Qi, Y. (eds) Plant Genome Editing with CRISPR Systems. Methods in Molecular Biology, vol 1917. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8991-1_2
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DOI: https://doi.org/10.1007/978-1-4939-8991-1_2
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-8990-4
Online ISBN: 978-1-4939-8991-1
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