Browsing by Author "Nimchuk, Zachary L."

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    CRISPR/Cas9-mediated resistance to cauliflower mosaic virus
    Liu, Haijie; Soyars, Cara L.; Li, Jianhui; Fei, Qili; Peterson, Brenda A.; Meyers, Blake C.; Nimchuk, Zachary L.; Wang, Xiaofeng (Wiley, 2018-02-06)
    Viral diseases are a leading cause of worldwide yield losses in crop production. Breeding of resistance genes (R gene) into elite crop cultivars has been the standard and most cost-effective practice. However, R gene-mediated resistance is limited by the available R genes within genetic resources and in many cases, by strain specificity. Therefore, it is important to generate new and broad-spectrum antiviral strategies. The CRISPR-Cas9 (clustered regularly interspaced palindromic repeat, CRISPR-associated) editing system has been employed to confer resistance to human viruses and several plant single-stranded DNA geminiviruses, pointing out the possible application of the CRISPR-Cas9 system for virus control. Here, we demonstrate that strong viral resistance to cauliflower mosaic virus (CaMV), a pararetrovirus with a double-stranded DNA genome, can be achieved through Cas9-mediated multiplex targeting of the viral coat protein sequence. We further show that small interfering RNAs (siRNA) are produced and mostly map to the 30 end of single-guide RNAs (sgRNA), although very low levels of siRNAs map to the spacer region as well. However, these siRNAs are not responsible for the inhibited CaMV infection because there is no resistance if Cas9 is not present. We have also observed edited viruses in systematically infected leaves in some transgenic plants, with short deletions or insertions consistent with Cas9-induced DNA breaks at the sgRNA target sites in coat protein coding sequence. These edited coat proteins, in most cases, led to earlier translation stop and thus, nonfunctional coat proteins. We also recovered wild-type CP sequence in these infected transgenic plants, suggesting these edited viral genomes were packaged by wild-type coat proteins. Our data demonstrate that the CRISPR-Cas9 system can be used for virus control against plant pararetroviruses with further modifications.
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    Genome-Wide Assessment of Efficiency and Specificity in CRISPR/Cas9 Mediated Multiple Site Targeting in Arabidopsis
    Peterson, Brenda A.; Haak, David C.; Nishimura, Marc T.; Teixeira, Paulo J. P. L.; James, Sean R.; Dangl, Jeffery L.; Nimchuk, Zachary L. (PLOS, 2016-09-13)
    Simultaneous multiplex mutation of large gene families using Cas9 has the potential to revolutionize agriculture and plant sciences. The targeting of multiple genomic sites at once raises concerns about the efficiency and specificity in targeting. The model Arabidopsis thaliana is widely used in basic plant research. Previous work has suggested that the Cas9 off-target rate in Arabidopsis is undetectable. Here we use deep sequencing on pooled plants simultaneously targeting 14 distinct genomic loci to demonstrate that multiplex targeting in Arabidopsis is highly specific to on-target sites with no detectable off-target events. In addition, chromosomal translocations are extremely rare. The high specificity of Cas9 in Arabidopsis makes this a reliable method for clean mutant generation with no need to enhance specificity or adopt alternate Cas9 variants.
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    SacB-SacR Gene Cassette As the Negative Selection Marker to Suppress Agrobacterium Overgrowth in Agrobacterium-Mediated Plant Transformation
    Liu, Yiming; Miao, Jiamin; Traore, Sy; Kong, Danyu; Liu, Yi; Zhang, Xunzhong; Nimchuk, Zachary L.; Liu, Zongrang; Zhao, Bingyu Y. (2016)
    Agrobacterium overgrowth is a common problem in Agrobacterium-mediated plant transformation. To suppress the Agrobacterium overgrowth, various antibiotics have been used during plant tissue culture steps. The antibiotics are expensive and may adversely affect plant cell differentiation and reduce plant transformation efficiency. The SacB-SacR proteins are toxic to most Agrobacterium tumefaciens strains when they are grown on culture medium supplemented with sucrose. Therefore, SacB-SacR genes can be used as negative selection markers to suppress the overgrowth of A. tumefaciens in the plant tissue culture process. We generated a mutant A. tumefaciens strain GV2260 (recA-SacB/R) that has the SacB-SacR cassette inserted into the bacterial genome at the recA gene locus. The mutant Agrobacterium strain is sensitive to sucrose but maintains its ability to transform plant cells in both transient and stable transformation assays. We demonstrated that the mutant strain GV2260 (recA-SacB/R) can be inhibited by sucrose that reduces the overgrowth of Agrobacterium and therefore improves the plant transformation efficiency. We employed GV2260 (recA-SacB/R) to generate stable transgenic N. benthamiana plants expressing a CRISPR-Cas9 for knocking out a WRKY transcription factor.