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bioRxiv · 10.1101/419952

Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis

Castel, B.·Tomlinson, L.·Locci, F.·Yang, Y.·Jones, J. D. G.
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Abstract

Bacterial CRISPR systems have been widely adopted to create operator-specified site-specific nucleases. Such nuclease action commonly results in loss-of-function alleles, facilitating functional analysis of genes and gene families We conducted a systematic comparison of components and T-DNA architectures for CRISPR-mediated gene editing in Arabidopsis, testing multiple promoters, terminators, sgRNA backbones and Cas9 alleles. We identified a T-DNA architecture that usually results in stable (i.e. homozygous) mutations in the first generation after transformation. Notably, the transcription of sgRNA and Cas9 in head-to-head divergent orientation usually resulted in highly active lines. Our Arabidopsis data may prove useful for optimization of CRISPR methods in other plants.

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Castel, B., Tomlinson, L., Locci, F., Yang, Y., Jones, J. D. G.. 2018-09-17. Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis. https://doi.org/10.1101/419952

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