New plant breeding techniques

Genome editing

Genome editing is a type of New Breeding Technique mediated by Programmable Sequence-Specific Nucleases (SSN). SSNs are enzymes that cause small breaks in DNA, which when repaired by the cell can produce mutations of various types depending on the molecular mechanism used in the repair. The particularity of the SSN applied to breeding resides in our capacity to program them with high precision, so that they land in specific sites of the plant genome and exercise their activity there. This, together with the abundant genomic and phenomic information generated in recent years in both model plants and crops, translates into an almost unlimited capacity for the generation of new genetic variability “directed” to areas of the genome previously identified by its functional interest. This new “directed” variability generated by SSNs can be used both to obtain new basic knowledge and to generate new traits of agronomic interest. In this second aspect, the range of uses and applications of SSN in the improvement of plants is huge, from breeding resistance to biotic and abiotic stresses, to increase productivity, improve nutritional content, etc.
In recent years, different types of programmable SSN have been discovered. SSNs based on proteins of the Zinc finger type (ZFNs) have been known since the 90’s decade in the last century. More recently, the SSNs based on TAL effectors were developed, which contributed to a qualitative leap in terms of efficiency compared to its predecessors. In both cases, the “programming” is based on the combination of several protein modules according to a previously elucidated code, which requires de novo modification of a multimodular protein for each genomic target. Although the development of new strategies for the synthesis and assembly of pieces of DNA has greatly facilitated the production of recombinant proteins, the programming of SSN with codes based on the combination of protein modules is laborious, expensive and not available for many laboratories.
The paradigm shift in the use of SSNs took place with the appearance of a new tool based on the acquired CRISPR bacterial immunity system, discovered and named by Francis Mojica from Alicante University in Spain. Unlike its predecessors, the programming of the CRISPR system does not require the de novo construction of multimodular proteins, but only the generation of a small RNA sequence complementary to the genomic target, which is incorporated into an invariable protein (Cas9) and serves as a guide (guide RNA or gRNA) to reach its genomic target. The simplicity of the CRISPR / Cas9 programming code greatly facilitates its design and construction, which makes it possible for many laboratories and end users to breed new specific “traits”. In addition, the CRISPR / Cas9 architecture allows the simultaneous programming of multiple objectives (a feature known as multiplexing), which could facilitate the accumulation of phenotypic characters, greatly accelerating improvement processes.
In general, there are three genetic editing strategies based on CRISPR / Cas9 according to the type of modifications introduced in the genome.

The most commonly used in plants is the so-called SSN-1 strategy, which consists of generating small random mutations (indels: insertions or deletions), as a consequence of DNA breakage in the target sequence and subsequent erroneous repair by the mechanism called NHEJ (Union of non-homologous ends). This strategy has proven to be very efficient in several species and aims to induce new characters by the loss of function of the mutated genes. For its part, the strategy known as SSN-2 offers greater precision, since it literally consists of rewriting a small genomic region that incorporates predefined changes that are encrypted in a “donor” DNA sequence that is transfers to the genome along with the SSN. For this, an alternative type of cellular repair, called Homologous Recombination, is used that repairs the genome using an external DNA template. Finally, the SSN-3 strategy is similar to the SSN-2, but its objective is not to introduce small “repairs”, but to incorporate “new genetic information” in precise genome sites taking advantage of the permissiveness of HR repair to accept donor sequences that they contain non-homologous regions in their central region. Both the SSN-2 and the SSN-3 (also known collectively as genetic targeting strategies or GT) extend the range of genetic variability to improve functions, providing a qualitative leap in the type of traits that can be obtained through these strategies. However, until recently, strategies based on homologous recombination have been inefficient in plants, which has meant that many of the approaches tested to date are of the “loss of function” type (SSN-1).
In all cases, to carry out the genomic editing, it is essential to transfer the SSN enzyme (for example, Cas9) to the nucleus of a plant cell, together with the gRNA and, when appropriate, the DNA of the donor containing the “new” “genetic sequence to be introduced (SSN-2 and SSN-3), to subsequently regenerate a complete plant from the cell or the edited cells. The most commonly used mechanism for the introduction of these elements into the cell is the genetic transformation mediated by Agrobacterium. The result is, transitorily, a transgenic plant that contains and expresses each and every one of the aforementioned elements. However, in a later step and in the case of plants that multiply sexually, the T-DNA can be eliminated by sexual segregation giving rise to non-transgenic plants that contains the desired genetic editing.
From the bioethical point of view and strictly attending to the final product, the SSN-1 and SSN-2 strategies generate plants that are practically indistinguishable from those generated by traditional breeding methods, and therefore it is argued that should not be considered as GMOs. For its part, the SSN-3 strategy would inevitably lead to GMOs, although with a greater degree of precision in the exact place of the genome in which the additional genetic information has been introduced.
Regarding the bioethical and regulatory implications, recently, alternative mechanisms have been successfully tested to directly introduce the ribonucleoprotein complex (Cas9 + RNAg) into the cell nucleus of protoplasts of different plant species, thus avoiding the use of intermediaries in the form of DNA. In this way, the editing process is completely assimilated to the process of chemical mutagenesis (the latter exempted from the regulation of OMG), so that the resulting plants can be marketed as non-transgenic.