CRISPR Towards a Sustainable Agriculture

Climate change and the need to feed an increasing population undermines food production and safety, representing the reasons behind the development of a new agriculture that is much more sustainable, productive and accessible worldwide. Genome editing and, in particular, clustered regularly interspaced palindromic repeats/CRISPR-associated protein (CRISPR/Cas) tools will play a major role in plant breeding to address these concerns. CRISPR/Cas includes a series of genome editing tools relying on the recognition and cleavage of target DNA/RNA sequences to introduce specific mutations.


Introduction
The term "sustainable agriculture" encloses practices of farming addressed to the production of highquality and safe agricultural products without compromising natural environments and the social and economic conditions of farmers. According to the Agricultural Sustainability Institute at UC Davis (https://www.nal.usda.gov/legacy/afsic/sustainable-agriculture-definitions-and-terms, accessed on 10 January 2022), the main goal of sustainable agriculture remains the preservation of the ability of future generations to meet their own needs, ensuring inclusive economic growth.
In the era of climate change, environmental threats will affect farmers at both the economic and the agronomic level, influencing crop yield and quality and with further negative effects on plant resistance to both abiotic and biotic stress. In order to counteract these environmental threats, and to have the chance to reach a sustainable production of industrial manufacture, new technologies will be used on the basis of the current knowledge in the biotechnology field.
Clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) represent a new perspective for genetic engineering and the last frontiers of the new breeding techniques (NBTs) and genome editing (GE) tools.

CRISPR/Cas System
CRISPR/Cas systems are part of the adaptative immune system of archaea and bacteria in ensuring protection against viruses. The mechanism of action relies on the recognition and the cleavage of foreign DNA or RNA of invading viruses. One of the main characteristics is the integration of short fragments of the invading DNA (spacers) into the CRISPR locus, conferring the heritable immunity of bacteria. The CRISPR locus consists of an array of unique spacer sequences, derived from foreign invading DNA and interspaced by identical repeat sequences (crisprRNA or crRNA), along with a sequence encoding for the trans-activating crRNA (tracrRNA) and a series of genes encoding CRISPR-associated (Cas) endonucleases, responsible for the cleavage of the genetic material. The transcription of the CRISPR locus allows for the formation of a single mRNA (pre-crRNA), which is partially complementary to tracrRNA, leading to the formation of an RNA duplex. The RNase III recognizes the RNA duplex and cleaves the double-stranded RNA (dsRNA) to form crRNA-tracrRNA complexes that activate and drive the Cas protein to the target sequence. The Cas protein is capable of introducing a double-strand break (DSB) only in the presence of a short conserved protospacer-adjacent motif (PAM) downstream of the target DNA, representing an essential prerequisite for the recognition of the target sequence .
CRISPR/Cas systems have been divided into two classes, six types and several subgroups based on Cas proteins and the nature of the interference complex . To date, the most common type, used as a genome editing tool, is the Type II Cas9 from Streptococcus pyogenes (SpCas9). The Cas9 protein Climate change and the need to feed an increasing population undermines food production and safety, representing the reasons behind the development of a new agriculture that is much more sustainable, productive and accessible worldwide. Genome editing and, in particular, clustered regularly interspaced palindromic repeats/CRISPR-associated protein (CRISPR/Cas) tools will play a major role in plant breeding to address these concerns. CRISPR/Cas includes a series of genome editing tools relying on the recognition and cleavage of target DNA/RNA sequences to introduce specific mutations.
consists of a bi-lobed architecture, including a large recognition (REC) lobe and a small nuclease (NUC) lobe. The NUC lobe includes a protospacer-adjacent motif (PAM)-interacting domain (PI) and two cleavage domains known as RuvC and HNH domains, each of which cleaves one strand of the target DNA three nucleotides upstream of the PAM sequence ( Figure 1A) . The REC lobe contributes toward activating Cas proteins when combined with the tracrRNA-crRNA complex.    Furthermore, the Cas9 protein has been re-engineered through point mutations in RuvC and/or HNH nuclease domains, inactivating the catalytic activity of each domain ( Figure 1A). It can be exploited to produce a nickase (nCas9) or a dead enzyme (dCas9) with complete loss of DNA cleavage activity. The nCas9 is usually used to enhance the specificity of CRISPR/Cas9, combining two nCas9 with pairs of sgRNAs that respectively cut only one DNA strand and therefore increase the number of recognized target bases. The dCas9 protein can operate as cargo to load and deliver proteins with different functions to a specific target site. The use of inactive enzymes (nCas9 or dCas9) can also facilitate the directional introduction of DNA fragments at a specific site and the assembly of base and prime editors (discussed below). This enzyme has been employed in base editing as well as in genetic and epigenetic regulation of gene expression . Otherwise, the most frequent use of dCas9 is in the activation (CRISPRa, activator) and repression (CRISPRi, interfering) of gene expression without introducing mutations in the genome [9] [10]

CRISPR Variants, Orthologs and Engineered Systems
[11] [12] [13] [14] [15] [16] ( Figure 1A and Figure 2). Following this approach, the dCas9, guided by a sgRNA to a specific regulatory region, is fused to transcriptional modulators, which are generally transcriptional factors or protein domains recruiting key regulatory elements to control gene expression. Their use has been reported in plants both as activators and as repressors . The dCas9, associated with an acetyltransferase or a methyltransferase, can also act at the epigenetic level as CRISPRi and CRISPRa systems .
Both nCas9 and dCas9 are involved in the assembly of base editing and prime editing systems. Base editors, including cytosine base editors (CBEs) and adenosine base editors (ABEs), catalyze C/G to T/A or A/T to G/C transitions in DNA or RNA molecules (Figure 1A and Figure 2). They have been optimized for plant genomes, providing high efficiency and precise editing at single base resolution . Base editing systems have been applied in major crops such as wheat, rice, maize, tomato, potato, soybean and rapeseed . Prime editing enables rewriting of genetic information into a specified DNA target site using a reverse transcriptase (RT) fused to a nickase enzyme and a prime editing guide RNA (pegRNA) to copy genetic information directly into the target genomic sequence (Figure 1A and Figure 2) .
Although prime editing still needs to be improved for editing efficiency in plants, it has been applied to obtain precise modifications in rice, maize, potato and tomato , especially using the second generation of prime editors (PE2), in which an engineered RT with improved features (such as increased processivity, substrate affinity and inactivated RNase H activity) is fused to an nCas9 .

New Perspectives for the Use of CRISPR/Cas System
CRISPR opens many doors for plant breeders to boost breeding programs towards ambitious targets, thanks not only to the feasibility of its application in a wide range of crop species but also to its versatility as a genetic tool that is constantly evolving. The use of CRISPR-engineered systems can foster the generation of a wide range of heritable genetic mutations such as In/Dels, targeted insertions, point mutations and nucleotide substitutions that are the most frequent modifications obtained, as well as targeted chromosomal rearrangements and genetic or epigenetic control of gene expression ( Figure 1A).
In addition, it offers the advantage of decreasing off-targets and pleiotropic effects, without neglecting the possibility of obtaining transgene-free edited plants.
The possibility of producing transgene-free plants has been exploited through self-pollination and segregation of exogenous DNA , transiently expressing a plasmid vector encoding for Cas9 and gRNAs or through the delivery of pre-assembled CRISPR/Cas9 ribonucleoproteins (RNPs) . By delivering the Cas9 protein instead of the vector, there is no transfer of specific DNA from one species to another. In addition, the Cas9 protein remains inside the cells for three/five days and then is degraded, also reducing the off-target events. All together, these approaches will make CRISPR a useful tool for a new generation of plants that potentially do not fall within the scope of the current regulation process of genetically modified (GM) products.
Moreover, the discovery and the improvement of CRISPR as a precise genome editing tool has resulted in the establishment of several CRISPR-based companies that are hoping to capitalize on this new technology. Indeed, the potential of gene editing to address the 21st century's problems has taken hold in the agricultural industry and the list of CRISPR companies is growing each day. As an example, Synthetic Here, researchers focus on the recent advances in CRISPR technology for the improvement of the most cultivated crop species and its potential applications in synthetic biology (Figure 2), with particular regard to traits such as quality, yield and stress tolerance. Lastly, researchers report a quick focus on the global regulatory framework on GM plant legislation.