Toolkits for GEaReD
For the succesfull implementation of GEaReD, technological improvements
at several stages are needed. 1) Efficient phenotyping technologies are
required. Wild plants can be more phenotypically diverse and they are
not well described in terms of agronomic traits compared to domesticated
crops. It is crucial that the right representatives with the desirable
traits within the species are selected for GEaReD. 2) DNA sequence
information of wild plants is also often sparse. Genome sequencing
efforts of candidate species such as wild relatives of current crops is
a prerequisite for releasing the full potential of GEaReD. The
combination of these improvements will lead to a further increase in
data and requires new data processing solutions. Artificial Intelligence
will be a key technology for this, and first generation AI is already
employed for genomic data processing. In the future, AI will further
support scientist with acquiring and connecting phenotyping data with
omic data enabling the construction of large databases. 3)
Transformation methods, usually involving tissue culture, are needed to
facilitate genome editing in candidate species. Even in many of our
crops, tissue culture technologies are limited to be efficient in only a
few cultivars of the crop. Ideally, there should be no such constraints
in a candidate species for GEaReD. 4) A platform of molecular tools for
precision genome editing over a wide range of species is essential for
releasing the full potential of the genome editing technologies. Among
these, the most frequently used tool is the CRISPR/Cas9 mediated genome
editing. However, recent progress has already provided many new
methodologies for targeted mutagenesis in the plant genome. First,
concurrent mutagenesis of multiple genes were made possible through
multiplex genome editing. This development was further accentuated
through the development of alternatives to the canonical PAM site (NCC).
Secondly, the function of the endonuclease were modulated to provide a
nickase activity that creates single strand breaks and allow site
specific genomic integration [10]. Base editing of specific
nucleotides is one of the latest developments. By introducing deaminases
or so-called base-editors, together with for example a nickase, specific
C-G base pairs can be changed to T-A base pairs and vice versa [11].
In another technology, the fusion of a transposase/recombinase to hijack
a transposon system, enables the introduction of sequences at a
predetermined location [11]. A similar approach to these transposon
systems, but more elegant way is prime-CRISPR. Hereby a reverse
transcriptase is fused to an endonuclease, enabling to integrate small
sequences at specific target sites in the genome [11]. These two new
techniques are perfectly suited to alter the activity of promoters, but
could also be used to re-design proteins. Changes in the amino acid
sequence could be used to alter activity, phosphorylation or
localization of proteins.