tRNA and ribozyme multiplexed guide delivery show different editing efficiencies in different cereal crops

Authors: MILNER, MATTHEW - US Department Of Agriculture (USDA); CRAZE, MELANIE - National Institute Of Agricultural Botany (NIAB); BATES, RUTH - National Institute Of Agricultural Botany (NIAB); WHITING, MICHELLE - National Institute Of Agricultural Botany (NIAB); BROOKS, JACK - National Institute Of Agricultural Botany (NIAB); MILLER, PETER - National Institute Of Agricultural Botany (NIAB); WALLINGTON, EMMA - National Institute Of Agricultural Botany (NIAB)

Submitted to: Frontiers in Plant Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/5/2024
Publication Date: 12/5/2024
Citation: Milner, M.J., Craze, M., Bates, R., Whiting, M., Brooks, J., Miller, P., Wallington, E.J. 2024. tRNA and ribozyme multiplexed guide delivery show different editing efficiencies in different cereal crops. Frontiers in Plant Biotechnology. 15:1426184. https://doi.org/10.3389/fpls.2024.1426184.
DOI: https://doi.org/10.3389/fpls.2024.1426184

Interpretive Summary: CRISPR/Cas9 technologies has opened a way in which scientists can understand the roles various genes in a genome play in plants as well as allow us to tailor specific version of genes to fit the diverse climates in which plants grow. Thus, an understanding of how to best deliver the components of the CRISPR/Cas9 machinery to plants in three important cereal species was examined to help researchers design their experiments effectively. What we found it the sequence we targeted itself was less important than the way in which the gRNA was delivered to the plant. Also of the various systems tested some worked equally well in rice but differently in wheat and barley suggesting that wheat and barley use a tRNA system to deliver the guides rather than a ribosome assembly.

Technical Abstract: With the cost and complexity of transforming crop species is high it is paramount to understand how the components needed to perform directed mutagenesis perform to achieve high rates of editing. Comparison of different guide systems targeting the genome using CRISPR/Cas9 in different cereal species highlights the need for a better understanding of components of genome editing which play a part in the targeted editing of important crop species. Here direct comparison of the same guide sequences driven by the same promoters in rice, wheat and barley shows large differences in editing efficiency. The differences seen were based on both the way in which the guides were expressed as well as factors outside of the guide sequence itself. While both the tRNA system and ribozyme system performed well in rice the same cannot be said about wheat and barley, where the tRNA system is better at achieving higher rates of editing in stable transformants. Overall high levels of editing can be seen in all three species when strong expression of the SpCas9 is coupled with the CmYLCV promoter to drive a tRNA of guides. Stable inheritance is also possible in all three species when plants are sampled early after the end of tissue culture. Overall inheritance was above 85% in all three species when mutations were identified early after plants coming out of tissue culture.