Crop Improvement Through CRISPR/Cas9 Gene Editing

Seshadri Dutta, NIIT University

Introduction

The global population is increasing at an alarming rate, with a quarter of the world’s population anticipated to reach 10 billion in the next 30 years. In the current situation, the most pressing problem facing humanity is ensuring food security for an expanding population. Extreme weather, decreasing agricultural land availability, and increased biotic and abiotic stressors, in addition to the expanding population rate, are important restrictions for farming and food production. With the advancement of technology that can aid in crop enhancement, productivity can be increased to some level. Creating differences in the gene pool is the most important prerequisite for producing novel plant varieties since genetic diversity is a significant source for trait improvement in plants.

Transgenes can be crossed out of the improved variety once the required changes have been made. However, when it comes to edible crop species, the integration of transgenes into the host genome is non-specific, often unstable, and a source of public concern. In the last few decades, genetic modification techniques such as physical, chemical, and biological mutagenesis have made significant contributions to understanding the role of genes and finding biological processes for crop improvement.

Genome editing is the process of changing a given genome directly and efficiently. There might be a variety of genes that can be changed in different cell types and species using nucleases that make specific changes. The application of genome editing methods utilizing site-specific nucleases (SSNs) has proven efficient gene editing in both animal and plant species during the last decade. These SSNs create double-stranded breaks (DSBs) in the target DNA, which are repaired through non-homologous end-joining (NHEJ) or homology-directed recombination (HDR) pathways, that result in insertion, deletion, and substitution mutations respectively in the target regions. Genome editing with engineered nucleases like zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has recently proven to be a viable tool for altering the targeted site in the genome and has been widely used in several crops.

CRISPR Technology

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in genome editing has initially proven its efficacy in mammalian cells in 2012 and this technology has transformed research in both animal and plant biology. The term CRISPR (first coined in 2002), refers to tandem repeats bordered by non-repetitive DNA regions that were initially identified downstream of Escherichia coli IAP genes. In 2005, these non-repetitive sequences were revealed to be homologous with foreign DNA sequences originating from plasmids and phages. Unlike ZFNs and TALENs, CRISPR genome editing is simpler, requiring the creation of a guide RNA (gRNA) of around 20 nucleotides that is complementary to the target gene’s DNA stretch.

The CRISPR cleavage methodology requires – a) a 20-nucleotide synthetic gRNA sequence that binds to the target DNA; b) The Cas9 nuclease enzyme that cleaves 3–4 bases after the protospacer adjacent motif (PAM). On the other hand, two domains make up the Cas9 nuclease, i) RuvC-like domains and ii) an HNH domain, each of which cuts one DNA strand. CRISPR/CAS9 mediated genome editing (CMGE) can be easily carried out in a small laboratory having sufficient basic plant transformation set. In plants, however, the majority of editing has been performed in model species such as Arabidopsis, rice, and tobacco, with just a few crop species having been studied utilizing CRISPR technology.

CRISPR for Crop Improvement

Just as in other animal model systems, Cas9 and sgRNA expression within targeted cells are sufficient to alter plant genomes. For expressing Cas9 or Cas9 variants and gRNAs in plant systems, there are numerous commercially available vectors. RNA polymerase III promoters that drive the production of short RNAs in their respective species, such as AtU6, TaU6, and others, control sgRNA. Cas9 is also positioned downstream of RNA polymerase II promoters, which direct the production of longer RNAs, similar to ubiquitin promoters. To target nuclear DNA, Cas9 is usually tagged with a nuclear localization sequence (NLS). The type of expression system to be worked on, the type of restriction sites present to insert sgRNA, and the type of Cas9 system all play a role in the vector selection.

So far, the CRISPR/Cas9 gene-editing technique has been used in almost 20 crop species for a variety of characteristics such as yield enhancement and biotic and abiotic stress management. Crop disease resistance and tolerance to significant abiotic stressors like drought and salinity have both been improved using CRISPR/Cas9-based genome editing. Although the majority of the studies were conducted in tomatoes, in 2014, the first CRISPR/Cas9-mediated genome editing in tomatoes was reported with the ARGONAUTE7 (SlAGO7) gene involved in leaf formation being targeted. The loss of function mutation of the SlAGO7 gene led to needle-like or wiry leaves, which were chosen as a target for the simple identification of altered plants. And interestingly the tomato plant showed early flowering as some of the developmental genes were also targeted and they showed a good mutation.

Case Studies:

1. Monocots: Rice (Oryza sativa)

Rice is a key staple food crop for more than half of the world’s population, and it is widely studied and used as a model crop for monocots owing to its smaller genome size. Several researchers have recently proven the use of CRISPR-based genome editing in rice, and a few studies have documented the use of genome editing to ameliorate biotic and abiotic stressors for rice crop development.

For the first time in any crop plant, scientists used both protoplast and particle bombarded rice calli systems to demonstrate sequence-specific CRISPR/Cas9 mediated genomic modification of three rice genes, phytoene desaturase (OsPDs), betaine aldehyde dehydrogenase (OsBADH2), and mitogen-activated protein kinase (OsMPK2) genes, which are involved in controlling responses to various abiotic stress stimuli. They developed two rice-specific genome editing vectors, pRGE3 and pRGE6, to show an RNA-guided genome editing method.

OsMPK5, a negative regulator of biotic and abiotic stressors in rice, was chosen for targeted mutagenesis and testing in rice protoplasts utilizing three gRNAs. A more accurate gRNA design technique resulted in a low level of off-targets. The study found that a cascade of sgRNAs did not affect the mutation rate of CRISPR/Cas9.

2. Dicots: Thale cress (Arabidopsis thaliana)

In 2013, Arabidopsis was the first plant to exhibit CRISPR/Cas9-based target genome editing. Brassinosteroid insensitive1(BRI1), jasmonate-Zim-domain protein 1(JAZ1), and gibberellic acid insensitive (GAI), three phenology-related Arabidopsis genes, were altered using the floral dip technique and genotyped using Restriction Fragment Length Polymorphism. The remarkable efficiency of mutation was confirmed by further sequencing. 

In protoplasts, CRISPR/Cas9 genome editing of five A. thaliana genes was investigated: PDS3 (PHYTOENE DESATURASE), AtFLS2 (FLAGELLIN SENSITIVE 2), CYCD3 (CYCLIN D-TYPE 3), and RACK1 (RECEPTOR FOR ACTIVATED C KINASE 1-AtRACK1b and AtRACK1c). Mutational efficiency varied and the efficacy of sgRNAs in causing gene editing effects was also established in this work.

Scientists used the 5′ regulatory sequences of three Arabidopsis genes (SPOROCYTELESS, DD45, and tomato LAT52) that target floral organs to induce Cas9 expression to create a germ-line-specific Cas9 system (GSC) for Arabidopsis. In the T2 generation, a considerable rise inheritable mutation rates, a decrease in chimera proportions, and an increase in mutation diversity were obtained, resulting in a specialized CRISPR/Cas9 system for genetic screening of fatal or other desirable mutations in Arabidopsis.

Applications and Concerns of CRISPR Technology

CRISPR/Cas9 technology is progressing at a breakneck speed. The majority of previous research has used gene knockout or gene silencing techniques which is a less precise but widely used approach. In mammalian and plant cells, gene knock-in or gene replacement methods that follow targeted mutagenesis through HDR showed encouraging outcomes. CRISPR/Cas9 technology is also being developed and used for a variety of reasons in functional genomics and molecular biology. The present focus is on individual gene loss-of-function and gain-of-function studies, as well as gene modules and genetic expression.

Many regulatory systems in many countries do not consider new breeding methods like ZFNs, TALENs, and CRISPR to be GMOs. But this system’s significance stems from its relative ease of use, great accuracy, and low initial cost. CRISPR technology’s main distinguishing characteristic, DNA cleavage detection via Watson and Crick base pairing, greatly simplifies DNA targeting. According to the United States Department of Agriculture (USDA), CRISPR/Cas9 altered crops can be produced and marketed without governmental oversight.

With the development and characterization of additional CRISPR effector proteins, the CRISPR/Cas system now has a wider variety of biotechnological uses. However, the CRISPR/Cas9 method has a number of drawbacks, including an inefficient HDR to NHEJ ratio and a limited number of simultaneous alterations per cell. Non-target effects are common with this technology, which makes it even more difficult to utilize.

Conclusion

CRISPR/Cas9-based genome editing is a game-changing technology. In the future, crop development using these techniques to boost yield, nutritional value, disease resistance, and other characteristics will be a major focus. It has been used extensively in various plant systems for functional research, fighting biotic and abiotic stressors, and improving other essential agronomic characteristics in the last five years. Though numerous changes to this technology are needed to increase on-target efficiency, the majority of the work done so far is preliminary and needs to be improved. Finally, because it has altered and metamorphosed our ability to modify and regulate prokaryotic and eukaryotic genomes, CRISPR-Cas9 technology has a promising future in producing the necessary mutation in plants. The extensive adoption of this technology will undoubtedly hasten its progress.

Also read: A photosynthesis model predicts 10-20% increase in crop yields

References:

1. Jaganathan, D., Ramasamy, K., Sellamuthu, G., Jayabalan, S., & Venkataraman, G. (2018). Crispr for crop improvement: An update review. Frontiers in Plant Science, 0. https://doi.org/10.3389/fpls.2018.00985
2. Karkute, S. G., Singh, A. K., Gupta, O. P., Singh, P. M., & Singh, B. (2017). Crispr/cas9 mediated genome engineering for improvement of horticultural crops. Frontiers in Plant Science, 0. https://doi.org/10.3389/fpls.2017.01635
3. Arora, L., & Narula, A. (2017). Gene editing and crop improvement using crispr-cas9 system. Frontiers in Plant Science, 0. https://doi.org/10.3389/fpls.2017.01932
4. Jia, H., Zhang, Y., Orbović, V., Xu, J., White, F. F., Jones, J. B., & Wang, N. (2017). Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker. Plant Biotechnology Journal, 15(7), 817–823. https://doi.org/10.1111/pbi.12677
5. Zaidi, S. S.-A., Mahas, A., Vanderschuren, H., & Mahfouz, M. M. (2020). Engineering crops of the future: CRISPR approaches to develop climate-resilient and disease-resistant plants. Genome Biology, 21(1), 289. https://doi.org/10.1186/s13059-020-02204-y
6. Wang, T., Zhang, H., & Zhu, H. (2019). CRISPR technology is revolutionizing the improvement of tomato and other fruit crops. Horticulture Research, 6(1), 1–13. https://doi.org/10.1038/s41438-019-0159-x

• The Corrosion Prediction from the Corrosion Product Performance
• Nitrogen Resilience in Waterlogged Soybean plants
• Cell Senescence in Type II Diabetes: Therapeutic Potential
• Transgene-Free Canker-Resistant Citrus sinensis with Cas12/RNP
• AI Literacy in Early Childhood Education: Challenges and Opportunities