Rapid Screening of CRISPR/Cas9-Induced Mutants Using the ACT-PCR Method

  • Chun Wang
  • Kejian WangEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1917)


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 



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.


  1. 1.
    Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821CrossRefGoogle Scholar
  2. 2.
    Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823CrossRefGoogle Scholar
  3. 3.
    Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826CrossRefGoogle Scholar
  4. 4.
    Hua Y, Wang C, Huang J et al (2017) A simple and efficient method for CRISPR/Cas9-induced mutant screening. J Genet Genomics 44:207–213CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Rice Biology, China National Rice Research InstituteChinese Academy of Agricultural SciencesHangzhouChina

Personalised recommendations