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Identification of CRISPR‐Cas9 Mutants in Arabidopsis Glutaredoxin Genes AtGRXS11 , AtGRXS6 , and AtGRXS3/4/5/7/8 G ene Cluster
Author(s) -
Fernandez Francisco,
Sanchez Kassandra,
Escobar Matthew
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.lb20
Subject(s) - glutaredoxin , crispr , biology , genetics , gene , arabidopsis , arabidopsis thaliana , agrobacterium , mutant , transgene , thioredoxin
Glutaredoxins are small redox enzymes that use glutathione as a substrate to reduce disulfide bonds in target proteins. Plants have a far greater number of glutaredoxins than other organisms, mostly due to a unique clade of class III glutaredoxins that is exclusively found in higher plants. Previous studies in our lab demonstrated that a small group of class III glutaredoxin genes are strongly upregulated by nitrate in Arabidopsis thaliana , and that RNA silencing of some of these glutaredoxins leads to increased primary root growth. Thus, glutaredoxins appear to link nutrient sensing with plant growth and root system architecture. To further explore this hypothesis, we are using CRISPR‐Cas9 technology to perform targeted mutagenesis of the AtGRXS11 , AtGRXS6 , and AtGRXS3/4/5/7/8 glutaredoxin genes in A. thaliana . CRISPR vectors have been assembled and utilized for Agrobacterium ‐mediated plant transformation. Transgenic plant lines were initially screened by restriction enzyme‐mediated cleavage of glutaredoxin sequences amplified from genomic DNA, demonstrating the loss of restriction sites near presumptive Cas9 cut sites. The presence of small insertion‐deletion mutations in the AtGRXS11 and AtGRXS6 genes was then verified by Sanger sequencing. The AtGRXS3/4/5/7/8 genes are located in a ~10 kb tandem array on A. thaliana chromosome 4, and we have attempted to delete this entire region using two CRISPR guide RNAs that target the flanks of this gene cluster. PCR screening has identified transgenic lines which appear to contain the targeted large‐scale deletion. Because the nitrate‐regulated glutaredoxins appear to be important regulators of primary root growth, this work could have significant broader implications related to important agricultural traits such as root depth (drought tolerance) and nitrogen use efficiency. Support or Funding Information This research was supported by NSF RUI‐IOS grant 1651584. We would like to thank the MARC U*STAR Program (NIH GM‐08807) for funding the student researchers Francisco Fernandez, Kassandra Sanchez This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .