Genome-editing applications in cereals.
Abstract
Recently developed methods for genome editing, representing a major breakthrough in the field of genetic engineering, will enable researchers to produce transgenic plants in a more convenient and safer way. Double-strand breaks (DSBs) are triggered by synthetic nucleases that later induce DNA repair mechanisms known as nonhomologous-end joining (NHEJ) or homology-directed repair (HDR) in the presence of a donor DNA. Gene targeting (GT) was earlier demonstrated in rice and maize genomes by exploiting several genes (Acetohydroxyacid synthase, waxy, ALS, OS11N3 etc.), while zinc finger nucleases (ZFNs) were used to modify IPK1 gene in maize. Clustered regularly interspaced short palindromic repeats (CRISPR-CAS) system has been shown to be efficient for targeted mutagenesis in wheat that has a hexaploid complex genome, rice, maize, and recently in barley. The CRISPR system is considered as advantageous over previous approaches due to its easy use and efficiency, however, needs to be improved for high off-target effects.
Keywords
- genome editing
- TALENs
- ZFNs
- CRISPR-CAS
- rice
- maize
- wheat
- barley
1. Introduction
Genome editing refers to the ability to perform controlled changes in the genome using specific nucleases. The ability of a recombination initiation by inducing double-strand breaks (DSBs) is a breakthrough in efficient genome editing and engineering of plants. Site-directed mutagenesis and gene replacement have been possible by these mechanisms that will lead to crop improvement and progress in functional genomics studies. Cereals, on the other hand, represent an important group in agriculture as those directly supply main carbohydrate sources for human food and animal feeding, e.g., rice for Asia, wheat for the whole world, and maize for the Americas. Grass family (known as
Genome modification studies have been launched in plants two decades ago with low-targeted integration frequencies [2]. By the discovery of nucleases inducing DSBs in specific loci, GT frequencies dramatically enhanced. In maize, acetohydroxyacid synthase genes (
By the advancement of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR-CAS) system, these technologies were performed in
Plant | Explant | Transformation method | Genome-editing approach | Gene/locus | Reference |
---|---|---|---|---|---|
Maize | Embryogenic cell cultures | Whisker-mediated | ZFNs | Shukla | |
Rice | Embryogenic cells | TALENs | Li | ||
Wheat | Cell suspensions | CRISPR | Upadhyay | ||
Rice | Callus | Particle bombardment | CRISPR | Miao | |
Maize | Callus | GT by HR | Ayar | ||
Barley | Immature embryos | CRISPR | Lawrenson | ||
Wheat | Protoplasts, immature embryos | Polyethylene glycol, particle bombardment | CRISPR | Zhang | |
Rice | Callus | CRISPR | Wang | ||
Maize | Protoplast callus | Polyethylene glycol, | CRISPR | Feng |
2. Principles of genome editing
The basis of genome editing relies on the formation of DSBs at specific loci and triggering DNA repair mechanisms. DSBs can be formed in eukaryotic cells by chemical and physical factors (reactive oxygen species, ionized-radiation, etc.) or by natural events like meiotic recombination. During the last decades, it was demonstrated that DSBs can be induced by synthetic nucleases and lately by the bacterial defence system CRISPR as well. A common feature of these synthetic nucleases is the combination of the bacterial type IIS restriction endonuclease
The strategies for genome editing are based on the endogenous cellular processes related to DNA repair and recombination. It is well known that recombination occurs naturally during meiosis and in many cases, involves chromatin exchange between homologous sequences. Such a recombination is designated as homologous recombination (HR) and is the governing recombination type and DNA repair mechanism in lower organisms such as bacteria, yeasts, and moss [17]. Homologous recombination frequency in lower organisms such as yeast and the moss
DNA repair through homologous recombination is designated as homology directed repair (HDR). This pathway is initiated by a DSB in DNA, which is a result of DNA damage or an endonuclease activity. In the presence of a donor DNA template and specific endonucleases, this pathway enables the replacement of a specific sequence. Therefore, one can use oligonucleotides such as triplex forming oligonucleotides (TFOs), short ssDNA or dsDNA donors such as T-DNA to induce HR. It should be noted that while T-DNA of
When looking at DNA repair mechanisms, a nonlegitimate recombination or nonhomologous-end joining (NHEJ) is the dominant repair pathway in higher organisms such as flowering plants and humans. In NHEJ pathway, two broken ends of DNA were ligated together without the need of a homologous template (Figure 1). This pathway can be looked on as an “SOS” pathway, where the cell is “panicked” and quickly repair the damaged DNA with putative errors in the process. The NHEJ pathway is usually recognized with many errors and, therefore, is an excellent choice for gene disruption. NHEJ can achieve all editing objectives, i.e., mutations including small deletions or insertions [22], as well as gene insertion and gene replacement [19, 23]. However, while getting a mutation is certain, the mutations are completely random, and unlike the homologous directed recombination (HDR), there is no way to predict which mutation will occur and what will be the final result.
Gene insertion is a combination of single DSB with a supplied donor DNA. Here, we mimic the T-DNA integration by
Gene replacement can be achieved by generating DSBs flanking the gene of interest and supplying a donor DNA. This may result in deletion and targeted insertion leads to a gene replacement [19].
In both NHEJ and HDR, the main challenge is screening and recognizing the relevant HDR event. When designing genome editing, these two DNA repair pathways (NHEJ and HDR) would be the main guidelines that should be considered. In general, sequence replacement can be achieved by HDR while mutations, deletions, and insertions can be achieved utilizing NHEJ (Figure 1).
3. Zinc finger nucleases (ZFNs)
Zinc finger nucleases (ZFNs) are synthetic endonucleases combining zinc finger DNA-binding domain with a nuclease subunit (typically
Major problems in using ZFNs are low specificity resulting in genotoxicity [27–30] and high complexity leading to low success rates of the designed enzymes [31].
DSBs by ZFNs have been applied mainly as a proof of concept and for research [32–34] in model plants and thus, all genome-editing strategies were explored by this pioneering system. In maize, inositol pentakisphosphate 2-kinase (
4. TAL effector proteins (TALENs)
Transcription activator-like effector nucleases (TALEN) are synthetic nucleases combining
TALENs where evolved from the
Similar to ZFNs, TALENs use
TALEN-directed mutations were generated in
5. CRISPR-CAS system
CRISPR or clustered regularly interspaced short palindromic repeats is a bacterial defence mechanism against bacteriophages. Although usually this system is composed of a cascade of many different proteins, in
The Cas9 has several functional characteristics including enabling binding of sgRNA, searching for complementary sequence, and cleaving the target sequence (HNH domain that cleaves the complementary DNA strand and RuvC domain cleaves the noncomplementary DNA strand). The most important is the PAM recognition domain that distinguishes the bacterial encoding RNA from the bacteriophage target sequence. In Cas9, this sequence requires NGG downstream to the targeted sequence. The Cas9 first binds the PAM sequence and then opens the DNA, allowing RNA/DNA hybridization or R-loop formation and then cleaves both DNA/RNA and ssDNA strands [38–40].
In 2013, several articles have been published reporting the plant genome engineering, using CRISPR system that five of them resulted in the generation of mutant plants with specific targeted loci [41]. Besides the applicability of CRISPR, the use of protoplast cultures for transient expression assays and agroinfiltration of leaf tissues have been preferred due to their advantages. Upadhyay
6. Conclusions and future perspectives
There have been substantial efforts to develop efficient technologies for GT in plants including cereals. For this aim, synthetic nucleases, including the mitochondrial
Frequency of HDR in plants (
There are several considerations and limitations for the major genome-editing technologies. TALENs are considered as the most precise genome-editing system for today. This suggests not only hitting the correct genomic location but more importantly less cytotoxicity from off-targeting effect. The high precision enables targeting multiple targets with confidence. High efficiency in genome editing is translated to the amount of screened plants in order to reach the desired modified plant. ZFNs are considered to be less efficient than TALENs and Cas9 shows higher efficiency. Both ZFNs and TALENs have to be redesigned for each target, while CRISPR-based methods require redesign of RNA molecules. Therefore, CRISPR methods are easier to employ and are more suitable for large genomic screenings or multiplex gene targeting. However, more evidence has raised Cas9 low specificity effect [47] and low homologous recombination ratio [48], whereas several approaches were taken to overcome these limitations of which dCas9-Fok might be the most promising [49–53], none addresses the size constrains presented by Cas9.
The huge size constrains of the current genome-editing tools prevent applying plant viral vector as genome-editing tool, thus the researcher should use meganucleases for these applications. In general, Cas9 and its derivative technologies would be sufficient for research of
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