5. CRISPR to modify the root system architecture
Genome editing – also known as gene editing – can be defined as a set of methods that enables targeted genome alterations. Over the past few years, this field has rapidly developed and proved promising in various areas, including basic biomedical research, medicine, and applied biotechnology. Three central platforms based on sequence-specific nucleases (SSNs) are currently in use: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided CRISPR (cluster of regularly interspaced palindromic repeats)–Cas (CRISPR-associated protein) nucleases (RGENs).
Sequence-specific nucleases operate by inducing double-strand breaks (DSBs) in site-specific chromosomal positions. The cellular DNA repair mechanism is stimulated as soon as a double-strand break occurs, activating two main groups of repair pathways: either one that needs a homologous sequence as a template (homology-directed repair, HDR) or one that has no or little requirement for sequence homology (non-homologous end-joining, NHEJ, and microhomology-mediated end-joining, MMEJ, respectively). The homology-directed repair relies on copying the target-homologous donor template to achieve accurate gene/sequence replacement. The NHEJ pathway is error-prone and often leads to nucleotide insertions and/or deletions (INDELs) and substitutions. MMEJ can, in the presence of a target-specific repair DNA, introduce insertions or otherwise produce specific deletions.
Methods using ZFNs and TALENs, first-generation genome editing tools, were first published in 1996 and 2010, respectively. Both involve artificial fusion proteins consisting of a sequence-specific DNA-binding domain (DBD) fused to the non-specific DNA cleavage domain of the restriction endonuclease Fok I. These two technologies have been employed to alter endogenous genes in more than forty organisms, even though protein design hinders their broader adoption.
Only three years after the TALE recognition code had been deciphered, the CRISPR-Cas9 technology entered the scene, revolutionising biological research. This gene-editing tool originated from the type II CRISPR-Cas system, an adaptive immunity system of bacteria and archaea that protects against viruses and plasmids. Its power lies in its simplicity: only two components, namely a single guide RNA (sgRNA) and the Cas9 endonuclease are in principle required to target any DNA sequence in any organism.
The defence process integrates short segments of invading DNA (called ”spacers”) into the bacterial genome, between copies of identical repeats; the resulting locus has been named CRISPR. Upon successive invasions, the CRISPR array is transcribed as precursor CRISPR RNA (pre-crRNA), processed to form a mature crRNA specific to the target sequence. The trans-activating crRNA (tracrRNA), target-independent, is also transcribed and promotes pre-crRNA maturation. The tracrRNA pairs with the repeat sequence of the crRNA and activate the Cas9 endonuclease, guiding it to the target sequence of the pathogen (named ”protospacer”). A prerequisite for cleavage is the presence of the protospacer adjacent motif (PAM), a 2-6 base pairs sequence downstream of the target, which discriminates between ”self” and ”non-self” DNA possible, thus avoiding autoimmunity. PAM recognition by Cas9 initiates DNA unwinding and base pairing between crRNA and protospacer, prompting Cas9 to generate a DSB three base pairs upstream of the PAM.
In 2012, Jinek and colleagues demonstrated that changing 20 nucleotides in the crRNA made it possible to reprogram the target DNA sequence. The crRNA:tracrRNA complex could be engineered in a chimeric single guide RNA (sgRNA).
These findings enabled the transition of CRISPR-Cas9 from a biological system to a two-component genome editing tool: as mentioned above, it only consists of the Cas9 enzyme, which cleaves the DNA three base pairs upstream the PAM, and a sgRNA, that guides the nuclease to the target sequence.  To overcome the target site limitation depending on PAM, several Cas variants from different organisms and after targeted evolution had been described and used in several plant species in recent years. Tools for the base and prime editing without double strand-break induction were developed. These systems allow the defined replacement of single nucleotides or short stretches of a genomic sequence.
Compared to first-generation platforms, the CRISPR-Cas9 technology offers many significant improvements. For example, while ZFNs and TALENs require the reengineering of the nuclease for each target sequence, the Cas9 protein is identical for all applications, so it is possible to address any genomic target by changing the sequence of the sgRNA following Watson-Crick base-pairing rules. Furthermore, designing and engineering guide RNAs is relatively simple, fast and low-priced, in contrast to the laborious and more expensive process demanded ZFNs and TALENs production. Another advantage of the CRISPR-Cas9 system is the possibility of using multiple sgRNAs with different sequences to target more than one locus simultaneously.