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.