Genome editing is a relevant, versatile, and preferred tool for crop improvement, as well as for functional genomics. In this update, I summarize the advances in gene-editing techniques, such as zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) associated with the Cas-9 and Cpf-1 proteins. These tools support great opportunities for the future development of plant science and rapid remodeling of crops. Among the various genome-editing tools, CRISPR has become the most popular. CRISPR has helped clarify the genomic structure and its role in plants: For example, the transcriptional control of Cas9 and Cpf1, genetic locus monitoring, the mechanism and control of promoter activity, and the alteration and detection of epigenetic behavior between single-nucleotide polymorphisms (SNPs) investigated based on genetic traits and related genome-wide studies. The present update describes how CRISPR/Cas-9 systems can play a valuable role in the characterization of the genomic re-arrangement and plant gene functions, as well as the improvement of the important traits of field crops with the greatest precision. In addition, the speed editing strategy of gene-family members was introduced to accelerate the applications of gene-editing systems to crop improvement. For this, the CRISPR technology has a valuable advantage that particularly holds the scientist’s mind, as it allows genome editing in multiple biological systems.
The rapidly growing population and a wide range of competitive dairy products and meat are pushing agricultural output and expanding the demand for feed, food, biofuels, and livestock . By 2050, the worldwide population will expand up to >9 billion, which may boost crop production demand by 100–110%. Consequently, the effective production of staple crops, such as Oryza sativa (rice), Triticum aestivum (wheat), Zea mays(maize), and Glycine max (soybean), will increase by just 38–67% [1,2]. Currently, numerous genome-editing tools and techniques have been adopted to overcome the problems arising in plants to compensate for the increased demand for food in the future . Gene-editing techniques, such as engineered endonucleases/meganucleases (EMNs), zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) , are important tools in plant research, as they allow the remodeling of future crops.
ZFNs were the first truly targeting protein reagents to revolutionize the genome manipulation area of research. ZFNs are binding domains for DNA that recognize three base pairs at the target site . ZFNs have been commonly used for targeted genome modification in different plant species, such as Arabidopsis thaliana (Arabidopsis), Nicotiana tabacum (tobacco), and maize [6–8]. Another site-driven mutagenesis genome-editing system, TALENs, was defined first in plant pathogenic bacteria (Xanthomonas) and is based on a concept similar to that of ZFNs. TALENs target one nucleotide at the target site (instead of three), thus rendering TALENs precise . TALENs were successfully used for genome editing in angiosperms and bryophytes [10,11].
Extensive investigation in this field led to the development of new genome-editing tools, such as CRISPR/Cas9 and CRISPR/Cpf1 [12,13]. Initially, these techniques were developed in prokaryotes, because there were no efficient genome-editing techniques for eukaryotes at specific sites. However, at the advent of eukaryotic genome editing, the CRISPR technology has revolutionized our ability to generate specific changes in crops . The CRISPR system requires only the guide RNA sequence to be changed for each DNA target site. Under different circumstances, the usage and modification of CRISPR technology are quite simple and efficient [15,16]. In this review, we highlight the use of genome-editing techniques to achieve highly precise and desired modifications in plants, as well as examples of the application of EMNs, ZFNs, TALENs, and CRISPR/Cas9/Cpf1 in various plants (Figure 1).
This genome-editing technique, which relies on the activity of RNA-guided nucleases and their mode of action, has gained much attention because of its versatility, potency, adequacy, and simplicity. The CRISPR/Cas9 system is a highly conserved system that originated from the bacterial species Streptococcus pyogenes. Its discovery was a significant breakthrough of the 20th century, as it represented an entirely distinct and divergent tool that was quickly examined by many bioinformaticians, biotechnologists [12,13], and microbiologists. In the 2012–2013 period, the CRISPR/Cas9 system was successfully implemented with remarkable cutting efficiency and simplicity to modify animal and plant genes. Studies reported three CRISPR/Cas systems (I, II, and III), each of which has distinct molecular mechanisms for nucleic acid piercing and targeting. The initial identification of Cas9 (formerly known as COG3513, Csx12, Cas5, or Csn1) through bioinformatics analyses revealed that it acts as a large multifunctional protein structure that comprises two nuclease domains, HNH and RuvC-like. The development of the CRISPR system proved to be advantageous for the manipulation of genetically modified cells in living organisms, as well as in culture.
Because of its versatility, simplicity, efficacy, and wide range of applications, the CRISPR/Cas9 system has been applied in many fields of research, such as biotechnology, genetic engineering, and fundamental and applied biology. With the expansion of the plant genome-editing system, the expression cassette of CRISPR/Cas9 [12,13] is transformed into the cells, incorporated into the nuclear genome, and expressed, followed by the cleavage of its target DNA sequence, usually 3 bp upstream of the protospacer adjacent motif (PAM) site. Double-stranded breakage of DNA activates two separate mechanisms of DNA repair, NHEJ and homology-directed repair (HDR). In the absence of a homologous template, NHEJ mediates the direct re-ligation of the broken DNA molecules, normally leading to insertions and deletions (InDels), or substitutions at the DSB site. However, in the presence of a donor DNA sequence, HDR may add new alleles, correct existing changes, or insert new sequences of interest. Although DNA becomes integrated into the plant genomic site at a low frequency, the integrated transgene can still be expressed and becomes functional only for a short period. Therefore, the expression of CRISPR/Cas9 via transgenesis may offer an alternative method for genome editing in plants. Interestingly, two simple and effective methods adopted for genome editing rely on the expression profile of the CRISPR/Cas9 DNA or RNA. For these methodologies, antibiotic and herbicide selection steps are adopted during post-transformation tissue culture and obstructed, which yield plants that regenerate from the induction cells of the callus that functionally express the CRISPR/Cas9 system.
The adopted CRISPR system and its usage will promote the rapid progress of crop breeding and functional genomics. Recently, new and versatile breeding technologies have been implemented to facilitate the engineering of multiple genetic loci in different breeding varieties, which will improve food security and strengthen crop amelioration [3, 5]. Moreover, the perusal of the literature for genomic sequences and their functions is a prerequisite for efficient genome editing. In the future, we will likely witness the increased use of CRISPR for clarifying genomic structures and their role in plants, such as the transcriptional regulation of Cas9 and Cpf1, the monitoring of genetic loci and mechanisms, and the regulation of promoter activity. Moreover, it will also include the modification and identification of epigenetic behavior in communicating the stable relationships between single-nucleotide polymorphisms (SNPs), which are investigated by genetic traits, and genome-wide association studies. Interestingly, this technique was designed to achieve phenotypic characterization in the T0 generation by engineering a genome-wide mutant library in rice. Upon consideration of the highly efficient editing achieved in the T0 generation, CRISPR/Cas9 was used to engineer a genome-wide mutant library in rapeseed, which will promote gene characterization and its beneficial applications at a later stage.
The CRISPR technology can classify any new crop traits in the category of plant synthetic biology. In our opinion, the saturation mutagenesis induced by CRISPR could be used to develop any desired plant protein when a proper selection tool is available. The use of this “faster and cheaper” method of evolution to optimize the role of metabolic enzymes in traits, such as crop production, quality, and disease resistance, should accelerate crop development; however, the CRISPR-associated technology would need to be strengthened. For example, improvements in the transformation methods and delivery of CRISPR/Cas agents to target cells will enable CRISPR in different tissues, including germline cells, and will increase the compatibility of plant species. However, the off-target issue is a big challenge in the application of gene-editing technology and a recent whole-genome sequencing analysis of CRISPR/Cas9-edited cotton plants revealed rare off-target mutations. The detailed strategy to increase on-target and reduce off-target effects of CRISPR/Cas9 was recently well reviewed. Targeted genome editing in rice using chemically modified donor DNA, which are designed for UTR or prompter region and the homology-directed repair method, was successful and further improvements are expected in the future. Creating a large population of CRISPR/Cas9-driven mutagenesis of promoters for developmental genes of tomato contributes to increased genetic variations. It is reasonable to expect that the development of various precision genome-editing technologies for targeted and precise gene/allele replacement, in combination with conventional breeding practices, will expedite the breeding of diverse elite crop varieties for the development of sustainable agriculture.
Genome editing is becoming the most used and versatile tool for crop improvement and functional genomics. The attractive survival landscapes, such as the efficiency, multiplexing, integrity, and simplicity, as well as the highly specific nature, of the genome-editing technologies mentioned here indicate the manner in which crop breeding is carried out and pave the way for plant breeding for the next generations. This new strategy for crop improvement has proven to be efficacious based on a review of the literature on transcriptomics, biotechnology, genomics, and phonemics. The regulation of transgenic crops was also coherently simplified to support the rapid progression of this technology and render these crops acceptable for consumer usage. In addition to these social and technical challenges, the CRISPR technology was used for the first time to edit plant genomes. Therefore, the use of genome editing on a large scale for crop improvement is already a reality. The journey of genome editing raises ethical questions that need to be addressed by researchers and society on a massive scale.
- Ray, D.K.; Mueller, N.D.; West, P.C.; Foley, J.A. Yield Trends Are Insufficient to Double Global Crop Production by 2050. PLoS ONE 2013, 8, e66428. [CrossRef] [PubMed]
- Röös, E.; Bajželj, B.; Smith, P.; Patel, M.; Little, D.; Garnett, T. Greedy or needy? Land use and climate impacts of food in 2050 under different livestock futures. Glob. Environ. Chang. 2017, 47, 1–2. [CrossRef]
- Zhang, H.; Zhang, J.; Lang, Z.; Botella, J.R.; Zhu, J.K. Genome Editing—Principles and Applications for Functional Genomics Research and Crop Improvement. CRC. Crit. Rev. Plant Sci. 2017, 36, 291–309. [CrossRef]
- Gaj, T.; Gersbach, C.A.; Barbas, C.F. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 2013, 31, 397–405. [CrossRef] [PubMed]
- Rai, K.M.; Ghose, K.; Rai, A.; Singh, H.; Srivastava, R.; Mendu, V. Genome engineering tools in plant synthetic biology. In Current Developments in Biotechnology and Bioengineering: Synthetic Biology. Cell Eng. Bioprocess. Technol. 2018. [CrossRef]
- Osakabe, K.; Osakabe, Y.; Toki, S. Site-directed mutagenesis in Arabidopsis using custom-designed zinc finger nucleases. Proc. Natl. Acad. Sci. USA 2010, 107, 12034–12039. [CrossRef] [PubMed]
- Petolino, J.F.; Worden, A.; Curlee, K.; Connell, J.; Moynahan, T.L.S.; Larsen, C.; Russell, S. Zinc finger nuclease-mediated transgene deletion. Plant Mol. Biol. 2010, 73, 617–628. [CrossRef]
- Shukla, V.K.; Doyon, Y.; Miller, J.C.; Dekelver, R.C.; Moehle, E.A.; Worden, S.E.; Mitchell, J.C.; Arnold, N.L.; Gopalan, S.; Meng, X.; et al. Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 2009, 459, 437–441. [CrossRef]
- Boch, J.; Scholze, H.; Schornack, S.; Landgraf, A.; Hahn, S.; Kay, S.; Lahaye, T.; Nickstadt, A.; Bonas, U. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 2009, 326, 1509–1512. [CrossRef]
- Li, T.; Liu, B.; Spalding, M.H.; Weeks, D.P.; Yang, B. High-efficiency TALEN-based gene editing produces disease-resistant rice. Nat. Biotechnol. 2012, 30, 390. [CrossRef]
- Kopischke, S.; Schüßler, E.; Althoff, F.; Zachgo, S. TALEN-mediated genome-editing approaches in the liverwort Marchantia polymorpha yield high efficiencies for targeted mutagenesis. Plant Methods 2017, 13, 20. [CrossRef] [PubMed]
- Bortesi, L.; Fischer, R. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol. Adv. 2015, 33, 41–52. [CrossRef] [PubMed]
- Zhang, Y.; Pribil, M.; Palmgren, M.; Gao, C. A CRISPR way for accelerating improvement of food crops. Nat. Food 2020, 1, 200–205. [CrossRef]
- Butt, H.; Zaidi, S.S.A.; Hassan, N.; Mahfouz, M. CRISPR-Based Directed Evolution for Crop Improvement. Trends Biotechnol. 2020, 38, 236–240. [CrossRef] [PubMed]
- Puchta, H. Applying CRISPR/Cas for genome engineering in plants: The best is yet to come. Curr. Opin. Plant Biol. 2017, 36, 1–8. [CrossRef] [PubMed]
- Ahmar, S.; Gill, R.A.; Jung, K.H.; Faheem, A.; Qasim, M.U.; Mubeen, M.; Zhou, W. Conventional and molecular techniques from simple breeding to speed breeding in crop plants: Recent advances and future outlook. Int. J. Mol. Sci. 2020, 21, 2590. [CrossRef]
Mr. Courage Dele Famusiwa, is a Lecturer II in Skyline University Nigeria. He has M. Tech in Applied Biochemistry and Toxicology from Federal University of Technology, Akure.