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Early And Present Applications Of CRISPR/Cas9

Gene Editing, Plant Genomics, CRISPR, Microbiome, Genetic Engineer

Genome editing technologies are a type of genetic engineering leading to the targeted modification of the genome of interest via the insertion, deletion or replacement of specific DNA sequences [1]. Amongst these, CRISPR/Cas9 is certainly the most promising and plant researchers have quickly realised its importance as its use is applied to several plant species [2, 3].

Damiano Martignago is investigating CRISPR/Cas9 efficiency in wheat by producing wheat lines expressing a wheat-optimised Cas9 and testing the guide RNAs in vitro to then target specific genes showing recognisable phenotypes. At the 5th Plant Genomics and Gene Editing Congress, he presented his research developments in investigating the applications of CRISPR/Cas9 in both rice and wheat.

Past Work And Applications Of CRISPR/Cas9 In Rice

Rice needs some alterations in its photoperiodic pathway to successfully thrive in Europe. At the University of Milan, Damiano worked on rice adaptation in the old continent to improve rice cultivation. However, the lack of rice mutants available in Europe pushed his team to develop their own mutant using CRISPR/Cas9. Although its application was quite new at the time, it was proved to be very efficient in the case of two rice florigen that are consecutive and adjacent in the genome. At the University of Milan, they could exploit non-conserved sequences to show individual mutants and to develop double mutants by using conserved regions.

Using CRISPR/Cas9 In Wheat

Recently, at Rothamsted Research, Damiano and his team are attempting to apply similar genome editing advances to wheat. However, applying CRISPR/Cas9 in wheat is not as easy. In fact, regenerating plants from genome edited protoplast in wheat is far more complicated and time consuming than in rice or other crops [1].

Wheat Vector

Figure 1. Vector structure optimised with RFP to C-terminus

They quickly understood if they wanted to attain better and more promising results, they had to build a wheat specific vector and remove any putative splicing site (Figure 1). To strengthen the vector structure the wheat codon was also optimised and a red fluorescein protein was attached at its C-terminal and a specific RNA expression cassette was also produced. This allowed an improved system for the exploitation of endogenous tRNA and the applications of eight guide RNA at a time to target potential conserved restriction sites.

To then verify the expression of the designed Cas9, wheat embryos were targeted with an instant IIB protein that marks the nuclear localisation, thus to confirm the presence of their engineered Cas9 in the nuclei of the embryos.

In future research, they are trying to extend these investigations to other plant species such as durum wheat, sorghum or barley trying to prove that efficiency of their designed Cas9 is reliable and can be also used in other systems other than rice and wheat.

View Damiano’s presentation slides here.

Reference:

  1. Martignago, D. (2017). CRISPR/Cas9 Technology In Rice And Wheat.
  2. Li, J.F., Norville, J.E., Aach, J., McCormack, M., Zhang, D., Bush, J., Church, G.M. and Sheen, J. (2013). Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature Biotechnology 31 (8), 688-691.
  3. Nekrasov, V., Staskawicz, B., Weigel, D., Jones, J.D. and Kamoun, S. (2013). Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nature Biotechnology 31 (8), 691-693.

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