Validation of sgRNA targeting for CRISPR/Cas9 editing in Brachypodium distachyon.

Crynodeb

Genome editing techniques have been in the spotlight for several years due to the potential they have in research and crop development and the uncertainty about the possible regulatory frameworks necessary for their use as a crop development tool. CRISPR/Cas9 has been the most prominent of all these tools, given the simplicity of the system and the high accuracy and efficiency when inducing targeted modifications in a specific sequence. Along the years, many
improvements have been achieved in the use of CRISPR/Cas9 as a lab tool, including optimisations in the system such as codon optimisation of the Cas9, sgRNA length and sequence optimisation and the use of different promoters for the expression of the editing machinery. At the same time, different crops have been edited using this tool. One of the most relevant clades commercially, the grasses, has been the focus of many studies reporting the use of CRISPR/Cas9.
Within this group, Brachypodium distachyon is rising as a prominent model due to its many advantages for its use in lab conditions when compared to other plants in the family. Although CRISPR/Cas9 has been used in this species, many of the optimisations previously mentioned are still to be achieved. With this context in mind, this project focused on the use of CRISPR/Cas9 in Brachypodium distachyon, finding new transformation methods for the introduction of the editing machinery in different tissues of the plant, including protoplasts and embryos, and investigating the possibility of using different promoters for the expression of the sgRNA. Additionally, the optimisation of the sgRNA sequence for its use in plants was assessed, proposing a new model for the prediction of editing efficiencies based on published sources. Finally, the regulatory context of edited crops was reviewed, focusing on the comparison of the possible outcomes obtained from different traditional breeding techniques and the possibility of the use of genome editing to replicate these outcomes. Finding possible equivalences between traditional breeding techniques and genome editing will help the case for an equivalent and proportional regulatory framework for the future

Dyddiad Dyfarnu	2022
Iaith wreiddiol	Saesneg
Sefydliad Dyfarnu	Prifysgol Aberystwyth
Goruchwyliwr	Huw Jones (Goruchwylydd) & Dylan Phillips (Goruchwylydd)