Biotechnol Ind J, Volume: 12( 12)
Crispr-Cas9: A Handy Tool, but Not in Genome-Editing Toolbox of All Plant Species
- Gurel E , Department of Biology, Abant Izzet Baysal University, 14280 Bolu, Turkey, Tel: +90 374 254 12 20; E-mail: [email protected]
Received: November 15, 2016; Accepted: November 17, 2016; Published: November 23, 2016
Genome modification/engineering, a tool for manipulation of organism’s genome using biotechnology, dates back to past century. Its resultant organisms are called genetically modified organism (GMO) in which recombinant nucleic acid (DNA or RNA) techniques are used. However, in genome editing as a newly developed subdivision of genome engineering, molecular scissors (engineered nucleases) are used to insert, remove, or replace target DNA, e.g. zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR–Cas9). Double-stranded breaks (DSBs) created at target sequence by activity of nucleases, will be repaired by either homologous recombination (HR) or no homologous end joining (NHEJ).
Function of CRISPRs and CRISPR-associated (Cas) proteins as a defense mechanism in prokaryotes was identified in 2000s [1,2]. About one decade later, its genome modification potential was realized , and growingly was employed in many phyla engineering. The reasons behind CRISPR/Cas9 prevalence over the last few years include easy to use, more versatile, cheaper, quicker and more precise unlike the other mentioned methods. For instance, CRISPR–Cas9 can be ordered at a cost about to 100 times cheaper than that of zinc finger. CRISPR/Cas9 target specificity is in a way that it cuts only certain target DNA sequences. Cas protein using CRISPR spacers is the cutter of given location. Its design, also, is not as complicated as ZFNs and TALENs.
The fundamental question, however, is raised on the possibility of successful delivery of CRISPR–Cas9 into many organism tissues besides model organisms, e.g. mice, fruit flies and arabidopsis. The advancement of improvement in agriculture is not very promising, hence it demands more attempt to keep pace with the most pressing problem, overpopulation of the world . To meet the challenges of agriculture, plant biologists believe that still the technical problems lying in plant genome modification are delivery systems of genome editors into plant cells, which then may face more difficulties in whole plant regeneration from some transformed cells.
Many plant species have been tissue cultured in vitro. For most of them, the technique is not efficient or transformed plant regeneration is very difficult. Although nearly all genome-editing methods can be applied in plant species, the major bottle-neck is transgene delivery into plant cells, and then the regeneration of transformants . This difficulty is more noticeable when one tries to transform different genotypes, where their response to the same method may differ significantly. The effort can be more tiresome if the genotype is easily regenerated but cannot be readily transformed, and vice versa.
Genotype-independent transformation and regeneration still is a goal. A few numbers of species are amenable to floral dip transformation , which sidesteps cumbersome tissue culture methods. A delivery method for many plants is mediated by Agrobacterium or other bacteria. However, this technique is species- or genotype-dependent. Another option for plant transgenesis and genome editing is biolistic method, which can be applied for many more plant species, but still suffer from the same problems stemming from tissue culture techniques. In addition, Agrobacterium enables researchers to generate single-copy insertions in comparison with multi-copy insertions when using biolistic methods. On the other hand, by using biolistic methods a clean gene technology can be expected , whereas Agrobacterium integrates its vector backbone sequences into host genome.
Even if both above-mentioned problems (i.e. delivery and regeneration techniques) are addressed, a new one may compound, that it is relatively large size of Cas coding sequence. Routinely, a plasmid in Agrobacterium shuttles Cas protein and its guidance RNA (sgRNA) genes into plant cells, which means the introduced genes into plant cells are normally integrated into the host genome. Researchers have recently adopted an alternative approach, direct protein delivery, enabling them to tackle the big size issue and at the same time, they would clear the hurdle imposed by current GMO regulations.
Recently, direct protein delivery, instead of DNA, into plant cells by biolistic method allowed a transient presence of the protein to modify genome in maize (Zea mays) . Similarly, Cas9 enzyme and sgRNA are firstly assembled outside a plant tissue, and then the complex, not the corresponding genes, is introduced into the plant. This method seems superior to clean gene delivery technology, since in addition to its improved efficiency, owing to transgene-free delivery of the complex into plant cells, the biosafety regulations on GMO can be removed.
A mini-Cas9 found in Staphylococcus aureus, 25% smaller than the conventional Cas9s, was used in mice for gene therapy. Using the mini-Cas9 is expected in plant genome editing as well. Inducing site-directed gene modifications in plants with no off-target effects is one of the areas researchers focus on .
Genome editing methods have been changing and improving constantly. One of the other methodological approaches is searching for new enzymes except Cas9 in various microbes with different properties. One of them is called Cpf1, which is smaller with different sequence requirements. It can efficiently cleave target DNA sequence immediately followed by a short 5′ T-rich protospacer adjacent motif (PAM), whereas a G-rich PAM follows the target in Cas9 systems. In addition, the targeted DNA is cleaved as a 5-nt staggered cut distal to the T-rich PAM . The other one called LshC2c2 from Leptotrichia shahii is specialized in targeting single-stranded RNA, while other ones target DNA . LshC2c2 application in plant sciences may help researchers in understanding and manipulation of plant transcriptomes and in counteracting virulent viruses having RNA as their genomes.
In conclusion, over growing population are seeking for more and better foods and feeds. Achieved progress in plant improvement does not seem sufficient. Newly discovered technologies can be employed to advance agricultural products to address consumer needs and to convince regulators. CRISPR/Cas9 aided genome editing technology has appeared very promising. Based on CRISPR/Cas system, many different methods and approaches have been tested so far . It has shown a wonderful and fast evolution from the date it was identified in a bacterium immune system to delivering with no transgene (CRISPR/Cas) integration into host genome . Moreover, a CRISPR-edited crop is likely to bypass USDA oversight ; and surprisingly, a genetically engineered mushroom has previously been free of USDA regulation . Despite the progress achieved in transgenesis, plant tissue culture still is very efficient and reliable technique for a few model plants. However, for many other major crops this technique is relatively applicable only for a few genotypes, which are not always commercial ones. Therefore, plant tissue culture lags behind transgenesis techniques, and still needs to be subjected to many experiments to keep apace with demand for secure food. CRISPR/Cas dawn promises a bright and sunny noon, if tissue culture clouds let it shine.
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