Genome Engineering for Crop Improvement. Группа авторов

and minerals to boost human health. The quality requirements vary around the globe as consumer preferences are variable depending on region and cultural values. The nutritional aspect is of unique importance as the nutrients are required in less quantity and almost two billion people across globe suffer from malnutrition, especially Zinc (Zn), Iron (Fe), Iodine (I) and vitamin A (Bhullar and Gruissem 2013). Analysis of grain quality attributes required much time and effort along with a great number of samples, mostly taken at the early stage of breeding and standard protocols must be followed (Cruz and Khush 2000).The latest accomplishments in the field of crop biotechnology enable to breakdown of information to understand the key enzymes playing their vital role in metabolic pathways, increasing the contents of required vitamins and reducing compounds, i.e. phytic acids and acrylamide‐forming amino acids. There are some cereals, i.e. Wheat, Maize and Rice which are biofortified to enhance food quality through fixing nutrition deficiencies. (Gil‐Humanes et al. 2014; Ye et al. 2000). A documented example is of Golden Rice, which is genetically engineered to markedly increase the β‐carotene to solve the problem of vitamin A deficiency (Ye et al. 2000). In this chapter, we have highlighted the application of targeted genome engineering approaches to understand and enhance the food quality with special emphasis on major crops. Based on previous findings, we have proposed the genome engineering techniques are an ultimate choice to enhance the food quality and ensure food security to the global human population.

3.2 Evolution and Historical Perspective of Genome Engineering

The utility of biological organism, procedures and techniques to develop products for improving human life is coined as genetic engineering/biotechnology. The natural variation has been taking place since the inception of plant kingdom, with natural selection mechanisms providing the means for plants survival under adverse situations. Moreover, humans have been utilizing the phenomena of artificial selection for around 10 000 years. Modern day crops are the outcome of drastic genetic alternations caused by artificial selection procedure. These genetic variations caused by natural mutation or artificial selection are important for any crop improvement program. The hypothesis gained more ground when it became evident that phenotypic changes can occur via induced mutations. Therefore, researchers utilized reagents, radiations and chemical mutagenic agents to generate mutation in targeted genome resulting in phenotypic changes (Shu et al. 2012). The mutation breeding concept conceived in the 1940s, revolutionized plant breeding ultimately leading toward the 1970s green revolution. A major breakthrough took place with the discovery of Agrobacterium tumefaciens utilized for carrying the gene of interest in a plant's genome producing transgenic plants (Nester 2014; Schubert and Williams 2006). The following technique carries several drawbacks, i.e. disruption of endogenous genes, random mutations, foreign DNA residues called genetically modified organisms (GMOs) and failure to utilize the native plant genetic repertoire. Keeping all these disadvantages in mind, there was a dire need to precisely and efficiently edit the genome of interest for the designated objectives of plant breeding.

In the 1980s, the first genome targeting system was introduced by Mario Capecchi along with the idea of double‐strand breaks (DSBs) for genome manipulations (Capecchi 1980), and later the ability to engineer genome by site‐specific DSBs was developed (Jasin 1996). After DSBs are generated, the cell's own repair machinery can be harvested to dictate the genetic outcome through the imprecise repair process of non‐homologous end joining (NHEJ) or the precise repair process of homology‐directed repair (HDR) (Schaart et al. 2016).The NHEJ mechanism possesses the ability to make frame shift mutation through the knockout of genes (Lloyd et al. 2012). With the ability to develop multiple DSB, it became possible to make further changes in the genome, i.e. chromosomal deletion, DSBs on different genome, chromosomal translocation and gene inversion (Ferguson and Alt 2001). In comparison to NHEJ, HDR methods are more efficient, producing the more precise repairs and enabling the sequence to be assembled as per user requirement (Puchta 2005). The HDR method can be efficiently utilized for precise genomic modifications allowing a variety of templates ranging from short oligonucleotides to generate DSBs for genome editing. Proteins that can been engineered and reprogrammed to bind and cleave DNA do not exist in nature. However, it is possible to program a DNA‐binding domain to bind to any user‐defined site‐specific sequence. This domain can be fused with another domain that can cleave the DNA specifically where it binds. These bi‐modular fusion proteins are the key to precise genome engineering because they can be programmed to bind to any user‐selected sequence and generate a DSB. Such programmable site‐specific binding proteins can carry other functional domains capable of effecting other genetic and genomic changes, including transcriptional regulation, epigenetic regulation, and even base editing without generation of DSBs (Komor et al. 2016). Complete genetic engineering events are well described. A genome‐engineering tool box has three major platforms: zinc‐finger nucleases (ZFNs), transcription activator‐like effector nucleases (TALENs), and CRISPR/Cas systems. ZFNs and TALENs are protein‐based and require protein engineering for every user‐defined sequence. However, CRISPR/Cas is an RNA‐guided system and can easily be engineered to bind to the DNA target (Quétier 2016). In the current genetic engineering era, the most widely utilized is CRISPR/Cas system which will be discussed in detail to achieve crops quality improvement.

3.3 CRISPR/Cas Genome Editing Systems

In the current scenario, CRISPR‐Cas9 is a widely adopted genome edited system (GET) due to simplicity, efficiency and versatility. In 1987, CRISPR array was identified in Escherichia coli (E. coli) genome (Ishino et al. 1987) with unknown biological properties. During 2005, several studies were successful that revealed a CRISPR array role in adaptive immunity based on the availability of homologs spacers to viral and plasmid sequence (Pourcel et al. 2005). Jinek et al. (Jinek et al. 2012) first reported an RNA‐guided DNA cleavage system with high required target efficiency. Deltcheva et al. (Deltcheva et al. 2011), uncovered CRISPR array provides protection against foreign DNA when coupled with Cas9 protein whereas, the immune system was based on RNA mediated DNA targeting. CRISPR/Cas systems are part of the adaptive immune system of bacteria and archaea, protecting them against invading nucleic acids (spacers) such as viruses by cleaving foreign DNA in a sequence dependent manner. The immunity is obtained via integration of spacers between two adjacent repeats at the proximal end of a CRISPR locus. The spacers are transcribed into CRISPR RNAs (crRNAs) approximately 40 nt in length by successive encounters with foreign DNA, and combines with trans activating CRISPR RNA (tracrRNA) to activate and guide Cas9 nuclease (Barrangou et al. 2007). The Cas9 cleaves the homologs double‐stranded DNA sequence (protospacer) in the foreign DNA. The availability of PAM downstream of the target DNA is a key for the successful cleavage with frequent 5′‐NGG‐3′ and less frequent 5′‐NAG‐3′ (Hsu et al. 2013). Seed sequence approximately 12 bp upstream of the PAM are integral for pairing between RNA and target DNA (Bortesi and Fischer 2015). Multiplex editing by the introduction of double‐stranded breaks (DSBs) on various sites and ability to edit several genes at the same time is a unique feature of CRISPR/Cas system (Zhou et al. 2014). It only requires monomeric Cas9 protein with sequence‐specific gRNA meanwhile, ZFNs and TALENs requires dimeric proteins with reference to target site. In the CRISPR/Cas system, the recognition of target site is based on the Watson and Crick model enabling the off‐target detection through sequencing analysis along with off‐target mutation can be fixed by careful design of gRNA. On the contrary, ZFNs and TALENs target specificity depends on protein‐DNA interaction with unpredictable and function‐specific properties (Cho et al. 2014). Moreover, the scientific community equipped with CRISPR expertise had contributed a lot to disseminate the fresh information to newcomers which is opposite to ZFN proprietary nature. Several online platforms are now available and are assisting researchers for all concerns related to CRISPR (Table 3.1; Figure 3.1).

3.4 Application of CRISPR/Cas System for Crops Quality Improvement

The improvement in quality is as important as yield and plant breeding programs are focusing on the improvement of grain quality. The grain quality is multigenic in nature and controlled by a