Investigation of DNA-free genome editing in Arabidopsis thaliana with pre-assembled CRISPR-Cas9 ribonucleoproteins and transcripts.

Thesis / 2017

Ellen Marie Klinkenberg

Programmable sequence-specific nucleases (SSNs) produces double stranded breaks (DSBs) in the genome in a site-specific manner. The following repair through endogenous repair systems allows targeted genome editing, which among others can be used to genetically improve crops. The superior SSN is the RNA-guided engineered nucleases (RGENs) derived from the adaptive immune system - clustered regulatory interspaced short palindromic repeats (CRISPR) and its associated protein 9 (Cas9) of Streptococcus pyogenes. Only three components are required to generate site specific cleavage; Cas9 endonuclease, a single guide RNA (sgRNA) containing spacer, and a genomic target site upstream of a protospacer adjacent motif (PAM). Upon binding of spacer to genomic target site, it directs cleavage of the site by Cas9 endonuclease. DNA-free genome edited plants are more likely to be labelled as non-gene modified organisms (non-GMOs), which will have a great impact and value for agriculture, and resulting in a more efficient breeding of crops and production of food. The main object of this thesis is to investigate DNA-free genome editing in Arabidopsis thaliana (A. thaliana) using RGENs, and further on investigate the ability to produce a DNA-free gene modified whole plant. To achieve this, we shall directly deliver the RGEN components as both pre-assembled ribonucleoprotein (RNP) complex and in vitro transcribed transcripts, to protoplasts of A. thaliana. The components to set up DNA-free RGENs genetic editing in protoplasts were successfully generated. Two sgRNA encoding gens, containing spacers targeting Phytoene desaturase 3 (PDS3) marker gene of A. thaliana was generated through sub cloning of spacers into a sgRNA expression vector, and success was confirmed by sequencing. Transcripts of the two sgRNA and Cas9 mRNA were generated by vitro transcription. To confirm transcripts functionality containing the predicted spacers, the sgRNAs and commercial Cas9 nuclease were combined to in vitro digest PDS3 target sites. This proved the functionality for one of the designed sgRNA. To be able to do in vivo editing, we needed to optimize the isolation of healthy protoplasts, which proved to be a challenge. High numbers of healthy protoplasts were isolated, but during transformation, they died. However, after substantial trials, and by changing several parameters we were able to optimize the protocol and achieved high number of healthy protoplasts both before and after transformation. Subsequently, isolated protoplasts were transfected with both pre-assembled ribonucleoprotein (RNP) complex and in vitro transcribed transcripts of Cas9 and sgRNA. Genetic modifications were analysed using T7 Endonuclease I assay, but without success. In order to be able to experimentally optimize our plans, we also successfully expressed the Cas9 protein, which can be purified and used for the pre-assembled RNP complex. Successful in vivo studies would help us comparing the editing efficiency of the pre-assembled RNP complex to Cas9 transcripts.