Skip to main content
SpringerLink
Log in

High efficiency of targeted mutagenesis in arabidopsis via meiotic promoter-driven expression of Cas9 endonuclease

  • Focus Article
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Key message

The use of a meiosis I-specific promoter increased the efficiency of targeted mutagenesis and will facilitate the manipulation of homologous recombination.

Abstract

The CRISPR/Cas9 system has been harnessed for targeted engineering of eukaryotic genomes, including plants; however, CRISPR/Cas9 efficiency varies considerably in different plant tissues and species. In Arabidopsis, the generation of homozygous or bi-allelic mutants in the first (T1) generation is inefficient. Here, we used specific promoters to drive the expression of Cas9 during meiosis to maximize the efficiency of recovering heritable mutants in T1 plants. Our data reveal that the use of a promoter active in meiosis I resulted in high-efficiency (28 %) recovery of targeted mutants in the T1 generation. Moreover, this method enabled efficient simultaneous targeting of three genes for mutagenesis. Taken together, our results show that the use of meiosis-specific promoters will improve methods for functional genomic analysis and studying the molecular underpinnings of homologous recombination.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  • Ali Z, Abulfaraj A, Idris A, Ali S, Tashkandi M, Mahfouz MM (2015) CRISPR/Cas9-mediated viral interference in plants. Genome Biol 16:238

    Article  PubMed  PubMed Central  Google Scholar 

  • Aouida M, Piatek MJ, Bangarusamy DK, Mahfouz MM (2014) Activities and specificities of homodimeric TALENs in Saccharomyces cerevisiae. Curr Genet 60:61–74

    Article  CAS  PubMed  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  CAS  PubMed  Google Scholar 

  • Fauser F, Schiml S, Puchta H (2014) Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J 79:348–359

    Article  CAS  PubMed  Google Scholar 

  • Feng Z, Mao Y, Xu N, Zhang B, Wei P, Yang DL, Wang Z, Zhang Z, Zheng R, Yang L, Zeng L, Liu X, Zhu JK (2014) Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA 111:4632–4637

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li J, Farmer AD, Lindquist IE, Dukowic-Schulze S, Mudge J, Li T, Retzel EF, Chen C (2012) Characterization of a set of novel meiotically-active promoters in Arabidopsis. BMC Plant Biol 12:104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mahfouz MM, Piatek A, Stewart CN Jr (2014) Genome engineering via TALENs and CRISPR/Cas9 systems: challenges and perspectives. Plant Biotechnol J 12:1006–1014

    Article  CAS  PubMed  Google Scholar 

  • Mao Y, Zhang Z, Feng Z, Wei P, Zhang H, Botella JR, Zhu JK (2016) Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis. Plant Biotechnol J 14:519–532

    Article  CAS  PubMed  Google Scholar 

  • Wang ZP, Xing HL, Dong L, Zhang HY, Han CY, Wang XC, Chen QJ (2015) Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol 16:144

    Article  PubMed  PubMed Central  Google Scholar 

  • Yan L, Wei S, Wu Y, Hu R, Li H, Yang W, Xie Q (2015) High-efficiency genome editing in Arabidopsis using YAO promoter-driven CRISPR/Cas9 system. Mol Plant 8:1820–1823

    Article  CAS  PubMed  Google Scholar 

  • Ye GN, Stone D, Pang SZ, Creely W, Gonzalez K, Hinchee M (1999) Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation. Plant J 19:249–257

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Qi-Jun Chen, State Key Laboratory of Plant Physiology and Biochemistry China Agricultural University, for providing the pHEE2A-TRI vector backbone. We thank members of the Laboratory for Genome Engineering at King Abdullah University of Science and Technology for helpful discussions and comments. The study was supported by the King Abdullah University of Science and Technology.

Author information

Authors and Affiliations

  1. Laboratory for Genome Engineering, Division of Environmental and Biological Sciences and Engineering, King Abdullah University of Science and Technology, 4700, Thuwal, 23955-6900, Saudi Arabia

    Ayman Eid, Zahir Ali & Magdy M. Mahfouz

Authors
  1. Ayman Eid
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zahir Ali
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Magdy M. Mahfouz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Magdy M. Mahfouz.

Ethics declarations

Conflict of interest

The authors declare no competing financial interests.

Additional information

Communicated by T. Cardi.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Eid, A., Ali, Z. & Mahfouz, M.M. High efficiency of targeted mutagenesis in arabidopsis via meiotic promoter-driven expression of Cas9 endonuclease. Plant Cell Rep 35, 1555–1558 (2016). https://doi.org/10.1007/s00299-016-2000-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-016-2000-4

Keywords

Search

Navigation