DNA-Free Genome Editing: Past, Present and Future

Abstract

Genome Editing using engineered endonuclease (GEEN) systems rapidly took over the field of plant science and plant breeding. So far, Genome Editing techniques have been applied in more than fifty different plants; including model species like Arabidopsis; main crops like rice, maize or wheat as well as economically less important crops like strawberry, peanut and cucumber. These techniques have been used for basic research as proof-of-concept or to investigate gene functions in most of its applications. However, several market-oriented traits have been addressed including enhanced agronomic characteristics, improved food and feed quality, increased tolerance to abiotic and biotic stress and herbicide tolerance. These technologies are evolving at a tearing pace and especially the field of CRISPR based Genome Editing is advancing incredibly fast. CRISPR-Systems derived from a multitude of bacterial species are being used for targeted Gene Editing and many modifications have already been applied to the existing CRISPR-Systems such as (i) alter their protospacer adjacent motif (ii) increase their specificity (iii) alter their ability to cut DNA and (iv) fuse them with additional proteins. Besides, the classical transformation system using Agrobacteria tumefaciens or Rhizobium rhizogenes, other transformation technologies have become available and additional methods are on its way to the plant sector. Some of them are utilizing solely proteins or protein-RNA complexes for transformation, making it possible to alter the genome without the use of recombinant DNA. Due to this, it is impossible that foreign DNA is being incorporated into the host genome. In this review we will present the recent developments and techniques in the field of DNA-free Genome Editing, its advantages and pitfalls and give a perspective on technologies which might be available in the future for targeted Genome Editing in plants. Furthermore, we will discuss these techniques in the light of existing- and potential future regulations.

Keywords: CRISPR/Cas; CRISPR/Cpf; DNA-free; Genome Editing; RGEN; RNPs; TALEN; plant.

FIGURE 1

Exemplary comparison of classic CRISPR/Cas9 and DNA-free CRISPR/Cas9. Comparison of classic CRISPR/Cas9 through the example of (A). tumefaciens transformation and DNA-free CRISPR/Cas9 exemplified by PEG mediated protoplast fusion. (A) In the classic CRISPR/Cas9 technique a T-Plasmid is designed that includes the desired gRNA and Cas9 coding sequences. Via Agrobacterium tumefaciens mediated transfer both gRNA and Cas9 sequences can be integrated in the host genome. In vivo gRNA and Cas9 are translated and the gRNA-Cas9 RNP complex is formed. Upon target detection, a double strand break is induced and mutations can arise by internal cell repair mechanisms. The CRISPR/Cas9 complex is constantly expressed and active in the cell. Finally, the genome can contain both the desired mutation and sequences for gRNA and Cas9. The transgene can be outcrossed but this is less practical or even impossible in vegetative propagated crops. (B) For DNA-free CRISPR/Cas9 recombinant Cas9 and in vitro translated gRNA are required. The RNP complex is formed in vitro and is directly delivered to protoplasts by e.g., PEG fusion. The complex is already active and can directly detect its target to induce double strand breaks. Cell repair mechanisms can lead to a mutated genome at the desired target without addition of any foreign DNA. The CRISPR/Cas9 complex is degraded within the cell and no longer available.