Crop improvement is essential to attaining world food security and enhancing nutrition for human beings. Both conventional breeding and modern molecular breeding have contributed to increased crop production and quality. However, the time and resources for breeding practices have been limited. It takes a long time to bring a novel improved crop to the market, and the genetic sources from wild species cannot be always available for crops of our interests. Genome editing technology implemented molecular breeding can overcome those limitations of time and resource by facilitating the specific editing of plant genomes. However, there is a long-lasting argument about the safety of genetically modified organisms (GMOs). In this review, we briefly summarize the principle of genome editing tools, focusing on the CRISPR/Cas9 system and the application of these tools to plants in the service of crop engineering.
The recent development of genome editing technology using programmable nucleases such as zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins (CRISPR/Cas) (Kim and Kim 2014) shed light on a new plant breeding approach; this technique can minimize the degree to which the target genome is genetically modified and can increase the specificity of the target locus (Shan
These genome editing technologies use the cell’s endogenous repair system when specific genomic regions are manipulated using sequence-specific nucleases (SSNs). SSNs can drive double-strand breaks (DSBs) in targeted sites of genomic DNA; those DSBs are repaired by processes known as non-homologous end-joining (NHEJ) (Rouet
The first- and second-generation system of SSNs, ZFNs and TALENs, are dependent upon proteins’ ability to recognize specific DNA sites and nuclease activity of FokI domains to cleave the target sequences (Kim and Kim 2014). ZFN is composed of 3–6 zinc finger proteins (ZFPs), and each of these proteins can recognize three base pairs of target DNA sequence. Two FokI nuclease domains attached to two subsets of ZFPs perform the DSBs. Like ZFNs, the TALEN system is composed of TALE DNA binding proteins, and each protein can recognize a specific single DNA base pair. Two FokI nuclease domains are attached to two subsets of TALE proteins to carry out DSBs at the target site.
More simply, the third-generation SSN system, CRISPR/Cas9, uses one protein for nuclease activity and a short single-strand guide RNA (sgRNA) sequence to guide this protein to the target sites (Fig. 1). In the bacteria’s immune system, captured small DNA fragments (~20 bp) from the foreign DNA of invading phages or plasmids are kept in the bacteria’s own genome; these fragments, known as protospacers, form a CRISPR (Makarova
For more detailed information about genome editing tools and comparisons of these tools, readers are encouraged to visit the brief history of CRISPR/Cas system (Doudna and Charpentier 2014; Hsu
One of the major goals of conventional plant breeding is to remove or add certain traits of crops to enhance their nutritional values or resistance to diverse biotic and abiotic stress (Allard 1999; Moose and Mumm 2008). For instance, the high level of erucic acids and glucosinolates in rapeseed was successfully removed by conventional breeding in 1970s, and now rapeseed has become the third most important source of vegetable oils in the world (Gupta and Pratap 2007). While this conventional breeding relies chiefly on natural variation in a gene of interest, physical or chemical mutagens have been used to generate random crop variants. In addition, the development of RNAi methods enables target genes to be silenced in specific tissues or at certain times, which results in the removal of unwanted traits from crops (Kusaba 2004; Tang and Galili 2004). However, RNAi-mediated gene silencing has had to overcome challenges from incomplete gene silencing, the co-silencing of unintended genes (off-targets), and the random integration of foreign DNA into plant genomes (if T-DNA harboring RNAi construct is transformed). Genome editing technologies can overcome some of these limitations of conventional breeding and RNAi-based approach to be used to generate improved crops indistinguishable from naturally occurring mutant crops.
Since the successful genome editing using CRISPR/Cas9 system in
Potato are normally harvested once a year and stored in a cold chamber to keep them fresh and prevent sprouting before cooking. During cold storage, starch in the potato tuber is degraded into glucose and fructose (Fig. 2A). Unfortunately, this cold-induced sweetening causes serious problems when potatoes are turned into potato chips or French fries; at high temperatures, the reduced sugars turn into dark-brown pigments and a strong carcinogen, acrylamide, forms during this process. Using a technique that relied on RNAi, the JR Simplot company developed the “innate potato” (Chawla
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Maize (corn) contains high levels of phosphorous, but most of phosphorous are stored in the form of phytic acid that is poorly digested in human. Mutant lines containing low levels of phytic acid were isolated by conventional breeding and mutagenesis, but it has proven difficult to introduce these mutations into different accessions of maize (Raboy 2007). Again, the RNAi technique has been used to silence the expression of phytic acid biosynthesis genes or transporter genes in maize (Raboy 2007). In 2009, Shukla
Finding caffeine-free coffee is the long-term goal of coffee breeders (Borrell 2012), because the process of removing caffeine from normal coffee beans usually costs a lot, sometimes produces toxic byproducts, and may reduce or remove other flavors. In 2003, Ogita
Genome editing techniques can generate targeted point mutations in crops. One of the earliest studies, ZFN-mediated genome editing, directed specific DNA sequences into the target locus and introduced a point mutation in the
Two research groups simultaneously proposed a possible strategy to make plants virus resistant using CRISPR/Cas9 technology (Baltes
A new RNA-guided genome engineering tool, the CRISPR/Cpf1 system, was reported to have properties different from those of the CRISPR/Cas9 system: the CRISPR/Cpf1 system has a single RNA-guided endonuclease lacking tracrRNA, 5′ T-rich PAM, and a 5-nt staggered DNA cut (Zetsche
As the CRISPR/Cas9 system consists simply of an sgRNA and a Cas9 protein, it can be assembled by
Recent advances in genome engineering have led us to a new era: crop genome editing will bring about the next “green revolution.” The CRISPR/Cas9 technique has led scientists and breeders to develop new strategies for crop improvement, which could provide sustainable solutions for the global food crisis. By domesticating wild plants, we have been able to generate high-yield crops and to produce staple foods. Besides of the high-yield production and staple foods development, we have met two concerns; both global environmental changes and reduced genetic diversity in ecological system. The new genome editing techniques with RNP-based transformation could pave the way for solving problems in food security, developing cultivars by which desired traits from a gene pool of wild species. Thus, novel and valuable plants generated by genome editing techniques can regain useful traits overlooked during domestication; these traits help plants survive unpredictable global environmental changes.