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

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

More Information

Corresponding author: E-mail address: wangkejian@caas.cn (Kejian Wang)

These authors contributed equally to this work.

摘要

- /
- /
- /
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.
注释:

HTML全文

参考文献(17)

[1]	Chenouard, V., Brusselle, L., Heslan, J.M. et al. A rapid and cost-effective method for genotyping genome-edited animals: a heteroduplex mobility assay using microfluidic capillary electrophoresis J. Genet. Genomics, 43 (2016),pp. 341-348
[2]	Cong, L., Ran, F.A., Cox, D. et al. Multiplex genome engineering using CRISPR/Cas systems Science, 339 (2013),pp. 819-823
[3]	Hu, X., Wang, C., Fu, Y. et al. Expanding the range of CRISPR/Cas9 genome editing in rice Mol. Plant, 9 (2016),pp. 943-945
[4]	Irion, U., Krauss, J., Nusslein-Volhard, C. Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system Development, 141 (2014),pp. 4827-4830
[5]	Kc, R., Srivastava, A., Wilkowski, J.M. et al. Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection Sci. Rep., 6 (2016),p. 32048
[6]	Li, J.F., Norville, J.E., Aach, J. et al. Nat. Biotechnol., 31 (2013),pp. 688-691
[7]	Ma, X., Chen, L., Zhu, Q. et al. Rapid decoding of sequence-specific nuclease-induced heterozygous and biallelic mutations by direct sequencing of PCR products Mol. Plant, 8 (2015),pp. 1285-1287
[8]	Montgomery, J., Wittwer, C.T., Palais, R. et al. Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis Nat. Protoc., 2 (2007),pp. 59-66
[9]	Ramlee, M.K., Yan, T., Cheung, A.M. et al. High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis Sci. Rep., 5 (2015),p. 15587
[10]	Sander, J.D., Joung, J.K. CRISPR-Cas systems for editing, regulating and targeting genomes Nat. Biotechnol., 32 (2014),pp. 347-355
[11]	Shan, Q., Wang, Y., Li, J. et al. Genome editing in rice and wheat using the CRISPR/Cas system Nat. Protoc., 9 (2014),pp. 2395-2410
[12]	Shan, Q., Wang, Y., Li, J. et al. Targeted genome modification of crop plants using a CRISPR-Cas system Nat. Biotechnol., 31 (2013),pp. 686-688
[13]	Shen, L., Hua, Y., Fu, Y. et al. Rapid generation of geneitc diversity by multiplex CRISPR/Cas9 genome editing in rice Sci. China Life Sci. (2017)
[14]	Thomas, H.R., Percival, S.M., Yoder, B.K. et al. High-throughput genome editing and phenotyping facilitated by high resolution melting curve analysis PLoS One, 9 (2014),p. e114632
[15]	Wang, C., Shen, L., Fu, Y. et al. A simple CRISPR/Cas9 system for multiplex genome editing in rice J. Genet. Genomics, 42 (2015),pp. 703-706
[16]	Yu, C., Zhang, Y., Yao, S. et al. A PCR based protocol for detecting indel mutations induced by TALENs and CRISPR/Cas9 in zebrafish PLoS One, 9 (2014),p. e98282
[17]	Zhu, X., Xu, Y., Yu, S. et al. An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system Sci. Rep., 4 (2014),p. 6420