OpenLB
Open Library of Bioscience
Advanced Search
Plant methods
2018;14 40.

A simple and cost-effective method for screening of CRISPR/Cas9-induced homozygous/biallelic mutants.

Jinggong Guo, Kun Li, Lifeng Jin, Rui Xu, Kaiting Miao, Fengbo Yang, Chaoya Qi, Lin Zhang, Jose R Botella, Ran Wang, Yuchen Miao
Author Information
  1. Jinggong Guo: 1State Key Laboratory of Cotton Biology, Department of Biology, Institute of Plant Stress Biology, Henan University, 85 Minglun Street, Kaifeng, 475001 China.
  2. Kun Li: 1State Key Laboratory of Cotton Biology, Department of Biology, Institute of Plant Stress Biology, Henan University, 85 Minglun Street, Kaifeng, 475001 China.
  3. Lifeng Jin: Zhengzhou Tabacco Research Institute of CNTC, No. 2 Fengyang Street, Zhengzhou, 450001 Henan China.
  4. Rui Xu: 1State Key Laboratory of Cotton Biology, Department of Biology, Institute of Plant Stress Biology, Henan University, 85 Minglun Street, Kaifeng, 475001 China.
  5. Kaiting Miao: 1State Key Laboratory of Cotton Biology, Department of Biology, Institute of Plant Stress Biology, Henan University, 85 Minglun Street, Kaifeng, 475001 China.
  6. Fengbo Yang: 1State Key Laboratory of Cotton Biology, Department of Biology, Institute of Plant Stress Biology, Henan University, 85 Minglun Street, Kaifeng, 475001 China.
  7. Chaoya Qi: Zhengzhou Tabacco Research Institute of CNTC, No. 2 Fengyang Street, Zhengzhou, 450001 Henan China.
  8. Lin Zhang: Zhengzhou Tabacco Research Institute of CNTC, No. 2 Fengyang Street, Zhengzhou, 450001 Henan China.
  9. Jose R Botella: 5School of Agriculture and Food Sciences, University of Queensland, Brisbane, QLD Australia.
  10. Ran Wang: Zhengzhou Tabacco Research Institute of CNTC, No. 2 Fengyang Street, Zhengzhou, 450001 Henan China.
  11. Yuchen Miao: 1State Key Laboratory of Cotton Biology, Department of Biology, Institute of Plant Stress Biology, Henan University, 85 Minglun Street, Kaifeng, 475001 China.
PMID: 29872452 DOI: 10.1186/s13007-018-0305-8

Abstract

BACKGROUND: The CRISPR/Cas9 system is being used for genome editing purposes by many research groups in multiple plant species. Traditional sequencing methods to identify homozygous mutants are time-consuming, laborious and expensive.
RESULTS: We have developed a method to screen CRISPR/Cas9-induced mutants through Mutation Sites Based Specific Primers Polymerase Chain Reaction (MSBSP-PCR). The MSBSP-PCR method was successfully used to identify homozygous/biallelic mutants in and , and we speculate that it can be used for the identification of CRISPR/Cas9-induced mutants in other plant species. Compared to traditional sequencing methods, MSBSP-PCR is simpler, faster and cheaper.
CONCLUSIONS: The MSBSP-PCR method is simple to implement and can save time and cost in the screening of CRISPR/Cas9-induced homozygous/biallelic mutants.

Keywords

Arabidopsis thaliana CRISPR/Cas9 Genome editing Mutant screening PCR Tobacco

References

Plant Cell Rep. 2015 Sep;34(9):1473-6 [PMID: 26082432]
Nat Biotechnol. 2013 Aug;31(8):686-8 [PMID: 23929338]
Science. 2011 Jul 15;333(6040):307 [PMID: 21700836]
Front Plant Sci. 2016 Apr 12;7:475 [PMID: 27148303]
Nat Biotechnol. 2013 Mar;31(3):230-2 [PMID: 23360966]
Science. 2009 Dec 11;326(5959):1501 [PMID: 19933106]
Plant Biotechnol J. 2014 Aug;12(6):797-807 [PMID: 24854982]
Sci Rep. 2015 Oct 26;5:15587 [PMID: 26498861]
Nucleic Acids Res. 2011 Jan;39(1):359-72 [PMID: 20699274]
Nat Commun. 2016 Aug 25;7:12617 [PMID: 27558837]
Methods. 2014 Aug 1;68(3):389-96 [PMID: 24780522]
Int J Mol Sci. 2015 Dec 21;16(12 ):30438-57 [PMID: 26703579]
Nat Rev Genet. 2010 Sep;11(9):636-46 [PMID: 20717154]
PLoS One. 2014 Dec 11;9(12):e114632 [PMID: 25503746]
Nat Methods. 2011 Jan;8(1):67-9 [PMID: 21151135]
Nat Protoc. 2007;2(1):59-66 [PMID: 17401339]
Nat Rev Mol Cell Biol. 2013 Jan;14(1):49-55 [PMID: 23169466]
Science. 2003 May 2;300(5620):764 [PMID: 12730594]
Science. 2003 May 2;300(5620):763 [PMID: 12730593]
Nat Biotechnol. 2013 Mar;31(3):227-9 [PMID: 23360964]
Genome Biol. 2015 Jul 21;16:144 [PMID: 26193878]
Front Plant Sci. 2017 Aug 03;8:1364 [PMID: 28824692]
Science. 2009 Dec 11;326(5959):1509-12 [PMID: 19933107]
Science. 2013 Feb 15;339(6121):819-23 [PMID: 23287718]
Plant Biotechnol J. 2016 Jan;14 (1):186-94 [PMID: 25867543]
Science. 2013 Feb 15;339(6121):823-6 [PMID: 23287722]
Sci Rep. 2014 Sep 19;4:6420 [PMID: 25236476]
Cell Mol Life Sci. 2017 Mar;74(6):1075-1093 [PMID: 27677493]
J Genet Genomics. 2017 Apr 20;44(4):207-213 [PMID: 28416245]
Journal Article

Word Cloud

Similar Articles

Cited By