Title : Targeted modification of the CENH3 gene in sunflower using a tomato yellow leaf curl (il-60-bs) viral vector system for the delivery of CRISPR/cas9 components
Abstract:
Doubled haploid (DH) technology could fast-track the production of elite inbred lines as DH lines contain completely homozygous genomes. However, the reliance of DH production methods on in vitro culture has resulted in the lack of a dependable DH production technique for sunflower. The identification of the centromere-specific histone 3 (CenH3) gene as a key player in chromosome segregation has been a breakthrough in eukaryotic haploid induction (HI) technology. In this study, we identified amino acids in the sunflower CenH3 gene that were previously associated with haploid induction in Arabidopsis thaliana. A virusbased delivery system for genome editing in sunflower was tested, with plants mechanically inoculated with a deactivated tomato yellow leaf curl virus (TYLCV) harbouring the Cas9 endonuclease, guide RNA and a sunflower CenH3-based donor “repair template”. PCR amplification of transformed sunflower cDNA confirmed Cas9 transcription in T0, T1 and T2 generations, indicating that the vector is actively expressing components, semi-persistently, and is seed transmissible. Two of the ten inoculated T0 plants contained mutations in the target area: One plant displayed some cells with full homology- directed repair (HDR) converting three amino acids (P51S, G52E, and A55V), while the other displayed partial HDR with only two amino acid conversions (P51S and A55V).This study demonstrates the upper limit of loading capacity of a geminiviral vector to be almost 8kb, while maintaining cell-to-cell movement and gene expression of the CRISPR components leading to successful gene editing in sunflower. This study provides a steppingstone in genome editing and potential trait improvement in sunflower.
Audience Take Away Notes:
- Genome editing is yet to be routine, the audience will learn a new technique for delivery of genomic material to tissue culture recalcitrant crops such as sunflower
- Efficient use and adoption of genome editing technologies
- This research could be used by other faculty to expand their own research or teaching
- This provides a practical solution to a problem that could simplify or make a designer’s job more efficient
- It will improve the accuracy of a design, or provide new information to assist in a design problem
