List of studies involving CRISPR/Cas9-mediated gene editing in grain crops.
The development of reliable and efficient techniques for making precise targeted changes in the genome of living organisms has been a long-standing objective of researchers throughout the world. In plants, different methods, each with several different variations, have been developed for this purpose, though many of them are hampered either by providing only temporary modification of gene function or unpredictable off-target results. The recent discovery of clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated 9 (Cas9) nucleases started a new era in genome editing. Basically, the CRISPR/Cas system is a natural immune response of prokaryotes to resist foreign genetic elements entering via plasmids and phages. Through this naturally occurring gene editing system, bacteria create DNA segments known as CRISPR arrays that allow them to “remember” foreign genetic material for protection against it and other similar sequences in the future. This system has now been adopted by researchers in laboratory to create a short guide RNA that binds to specific target sequences of DNA in eukaryotic genome, and the Cas9 enzyme cuts the DNA at the targeted location. Once cut, the cell’s endogenous DNA repair machinery is used to add, delete, or replace pieces of genetic material. Though CRISPR/Cas9 technology has been recently developed, it has started to be regularly used for gene editing in plants as well as animals to good success. It has been proved as an efficient transgene-free technique. A simple search on PubMed (NCBI) shows that among all plants, 80 different studies published since 2013 involved CRISPR/Cas9-mediated genome editing in rice. Of these, 20, 13, and 24 papers have been published in 2019, 2018, and 2017, respectively. Furthermore, 20 different studies published since 2014 utilized CRISPR/Cas9 system for gene editing in wheat, where five of these studies were published in 2019 and seven were published in 2018. Genomes of other grain crops edited through this technique include maize, sorghum, barley, etc. This indicates the high utility of this technique for gene editing in grain crops. Here we emphasize on CRISPR/Cas9-mediated gene editing in rice, wheat, and maize.
- gene editing
- cereal crops
Perhaps one of the most important differences that can define humans apart from others in the animal kingdom is the ability to make choices among good, better, and best. The ability to reason, argue, research, and adopt what is (or at least what
With further developments in genetic engineering, genome modification studies were performed in plants . This was specifically aided by the discovery of nuclease enzyme, the utilization of different bacterial plasmids for cloning of target genes in bacteria as well as target plants and by the development of plasmid vectors carrying antibiotic resistance genes, a variety of constitutive, inducible as well as tissue-specific promoters, and different reporter genes that made the cloning and transformation easier. The discovery of the Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more recently that of the clustered regularly interspaced short palindromic repeats (CRISPRs) in genomes has further aided the genome editing techniques in different plant species.
1.1 Genome editing in nature
Genome editing involves molecular tools that lead to targeted modification of specific DNA sequences inside the genome. These are usually based on the production of double strand brakes (DSBs) at the specific DNA sites that trigger DNA repair of the cell. In plant cells, this generally happens through the process of non-homologous end-joining commonly abbreviated as NHEJ. However, this process is error-prone, thus exploited by scientists to target genes for possible modification. Sometimes, plants repair the DSBs through homologous recombination which is error-free. Hence, it could be used by scientists to precisely edit DNA or insert DNA sequences in a given genome.
Genetic modification or genome editing is already in practice in cells of living organisms and is one of the most magnificent specters of nature. Natural DNA repair mechanisms, losing and acquiring of genetic material in bacteria, transmission of bacteriophages through bacterial transduction, and genetic modification of plant genome by Ti plasmid during infection by
In the last few decades by unraveling the details of mechanisms underlying natural genome editing, scientists have shown that such editing can also be induced by synthetic nucleases and more recently by the CRIPR system adopted from bacterial defense system which is one of the most promising gene editing technologies introduced in 2012.
1.2 The CRISPR/Cas system
Among the different types of defense systems found in prokaryotes and eukaryotes, a unique molecular system known as the CRISPR/Cas not only provides protection against genetic attack, but also keeps a record or genetic memory of such attacks for future safety. This technique has been employed in several different studies by scientists from around the world for targeted genetic modifications in a plethora of living organisms, but perhaps most ambitiously by the Chinese scientists who not only used the technique for gene editing an armada of plant and animal species but also humans (https://www.nature.com/articles/d41586-019-00673-1), which raises certain ethical concerns about the practical application and subsequent implications of such studies.
A key factor for sustainable agriculture is improvement of crops via genetic engineering. It is true that DNA-free editing techniques are now desirable for molecular breeding in crops for which CRISPR/Cas may offer a better option for more complex and controlled genetic rearrangements. CRISPR arrays are characterized by a series of 20- to 50-bp genomic loci, which are unique spacers separated by direct repeats that usually have similar length to preceding AT-rich fragments . CRISPR loci were discovered about two decades ago in
Though CRISPR/Cas9 technology is relatively recent, it has started to be regularly used for gene editing in plants as well as animals to good success. It has been proved to be an efficient transgene-free technique. A simple search on PubMed (NCBI) shows that among all plants, 80 different studies published since 2013 involved CRISPR/Cas9-mediated genome editing in rice. Of these, 20, 13, and 24 papers have been published in 2019, 2018, and 2017, respectively. Furthermore, 20 different studies published since 2014 utilized CRISPR/Cas9 system for gene editing in wheat, where five of these studies were published in 2019 and seven were published in 2018. Genomes of other grain crops edited through this technique include maize, sorghum, barley etc. This indicates the high utility of this technique for gene editing in grain crops. Evidence that CRISPR/Cas9 system can be used for gene editing came around 2013 [6, 7] and the next paper showing the use of this technique for gene editing in rice and wheat was published in 2014 . Furthermore, other studies published from 2013 to 2015 showed the use of this technique for gene editing in an array of organisms such as yeast, zebrafish, fruit flies, mosquitoes, nematodes, mice, monkeys, and human embryos, and several plant species, indicating sufficiently fast and easy application of this technique in this crop.
1.3 CRISPR/Cas9 for crop improvement in rice, wheat, and maize
The importance of agriculture for human survival cannot be questioned. As mentioned earlier, plants are the only primary producers on planet earth providing food, fiber, and other raw material generating the bulk of energy required for the growth of human population. However, plants/crops are facing several challenges and now their own survival may well be at stake. In this context, classical breeding methods may not be suitable for increasing per unit area production relatively quickly. Hence, the rational use of biotechnological tools is of paramount importance. Editing crop genomes is a promising technique to cope with agricultural challenges. Development of these methodologies is useful for genetic improvement of crops . In 2013, the pioneering works published by Lit et al. using
The maize ARGOS8 variants generated via CRISPR/Cas9 showed significantly improved yield under drought stress conditions .
Rice, given its worldwide relevance for food, has been one of the most studied crops in terms of CRISPR/Cas9 application [31, 32, 33, 34, 35, 36]. These include genetic modification for increased disease resistance , and herbicide resistance . At present, more than 80 different research papers have been published using CRISPR-based genetic editing in rice and more than 15 papers involving wheat. These studies involve applicative use of CRISPR in rice. Li et al.  developed photo- and thermo-sensitive male sterile rice lines using this technique to exploit heterosis and speed up breeding . Rice cultivars carrying high genetic resistance to the rice blast disease have been recently developed by Wang et al. . Li et al.  at the South China Normal University, China, targeted four yield-related genes
Both durum and bread wheat have also been the subject of successful CRISPR/Cas9-mediated genetic modification for powdery mildew resistance and other objectives [42, 46, 47, 48]. However, in wheat, regeneration of plants from CRISPR-edited protoplasts has been difficult . In addition, the complexity of wheat genome together with time-consuming tissue culture techniques has made it difficult for scientists to undergo ambitious genome editing projects via CRISPR/Cas9 in this important cereal. Researchers at the Chinese Academy of Science obtained the first CRISPR/Cas9-edited wheat plants  by editing three homoeoalleles of a hexaploid bread wheat cultivar to confer heritable resistance against powdery mildew of wheat. Later, the same research group obtained transgene-free genome-edited wheat plants using transient expression of CRISPR/Cas9 DNA or RNA . In 2018, Wang et al.  knocked out all the wheat homologs of the rice
In their book chapter, Chilcoat et al.  discuss the use of CRISPR/Cas9 for crop improvement in maize and soybean and have discussed the molecular details of gene editing projects via CRISPR/Cas9 such as those involving
Table 1 summarizes the list of important studies involving CRISPR/Cas9-mediated gene editing in grain crops.
|1||Rice blast fungus (||RNAP II|||
|3||MPK1 and MPK6|||
Conflict of interest
The authors declare no conflict of interest.