When many diplomates and other professionals are unable to address the direst problem of feeding 9.6 billion people by 2050, scientists are using advanced technologies to increase the productivity of the crops. Yes, scientists and plant breeders are working for ages to increase the productivity of plants either by traditional plant breeding methods or using QTL mapping techniques to identify the potential hybrid at the genomic level or by using gene editing. Nevertheless, the sad part is, Genetically modified crops are mostly not excepted over traditional domesticated varieties. Now it is time to create awareness among the people that employing gene-editing techniques such as CRISPR/Cas9 can solve the food crisis by increasing the resistance of crops towards climate change and diseases, leading to a rise in the plant’s yield.
Why CRISPR/Cas9 ?
Due to the increase in population and change in climatic conditions, food productivity increase by the green revolution is no longer effective. Furthermore, the use of conventional breeding methods demands much time, is laborious, and complicated. Thus, the shift from conventional plant breeding methods to much more modern, faster and specific with gene-editing techniques was required. Specifically, to achieve the aim of developing a gene pool that will survive both biotic and abiotic stress. Some of the sequence-specific techniques, such as zinc-finger nucleases (ZNFs), transcription activator-like effector nucleases (TALENs) and meganuclease, are efficient for plant-editing at specific locus but require complex protein engineering for construction limits their application.
Instead of laborious designing and expression of two different DNA binding domains essential for per target site (ZFNs and TALENs), the requirement is an 18-20bp oligonucleotide, which uses DNA-RNA interaction (CRISPR/Cas). Significantly, CRISPR/Cas precise site-directed editing is simple, flexible, cost-effective, and straightforward engineered nuclease. Hence it is widely used by the scientist for various agricultural applications.
CRISPR/Cas: Success stories of enhanced food production
Domestication of Wild tomato (Solanum pimpinellifolium)
Pic credit: Zsogon et al., 2018
The extensive focus on the yield through domestication in worldwide consumed vegetable fruit tomatoes (S. lycopersium) has reduced genetic diversity, nutritional value and taste. Zsogon et al., 2018 used multiplex CRISPR/Cas9 to edit six genes in a wild relative S. pimpinellifolium (pea-sized fruits), resulting in transformed fruit number (MULTIFLORA, (MULT)), shape (OVATE, (O)), size (FASCIATED (FAS) and FRUIT WEIGHT 2.2(FW 2.2)), nutrient content (LYCOPENE BETA CYCLASE (CycB)) and plant architecture (SELF- PRUNING (SP)) in a single generation to create a novel crop. In short, a construct of target-specific transformation vector harbouring six single gRNAs (guide RNAs) could edit four genes (Ovate, CycB, FW 2.2 and SP), where the loss of function in self-pruning results in compact architecture with reduced height and number of sympodial; while distinct homozygous deletions in the first exon of the ovate (O) gene gave elongated fruits with less rain-induced fruit cracking trait. Whereas in the second round of editing, there was loss-of-function MULT allele and biallelic deletions in exon one & a heterozygous deletion in exon 2 of the CLV3 gene (FAS phenotype) conferred a higher number of fruits with up to 200% increase in fruit weight as compared to S. pimpinellifolium. Furthermore, loss-of-function mutations in the first exon of CycB enhanced the lycopene content without negatively affecting the accumulation of β-carotene or lutein.
Disease resistance: Powdery Mildew resistance
The disease developed in wheat by biotrophic fungal pathogen Blumeria graminis f. sp. is responsible for decreasing production. The study showed that the TaEDR1 play a negative role in Mildew resistance, thus knock-down of all three homologous (TaEDR1-1A, TaEDR1-1B and TaEDR1-1D) TaEDR1 with CRISPR/Cas9 using highly-specific guide RNA single (conserved EDR1 N-terminal regulatory domain) confer the resistance. Thus, resistant Taedr1 wheat plants generated did not show mildew-induced cell death (Zhang et al., 2017)
Similarly, the SlMlo1 gene trimming with CRISPR-Cas9 resulted in resistance to Oidium neolycopersici causing Powdery mildew in tomatoes. Self-pollination was employed to ensure CRISPR-Cas9 free plant and this new non-transgenic plant variety named “Tomelo” by the author (Nekrasov et al., 2017).
Cold-resistant in Rice (Oryza sativa)
The seedlings of osmyb30-7, osmyb30-11 and WT after being treated in 4°C chamber. Credit Zeng et al., 2020.
CRISPR/Cas9 was used to mutate OsPIN5b, GS3, and OsMYB30 individually and simultaneously resulted in several transgenic lines such as nine transgenic lines of the ospin5b/gs3, six lines of ospin5b/osmyb30 and six lines of gs3/ osmyb30; that exhibited increased panicle length(ospin5b), enlarged grain size (gs3) and increased cold tolerance (osmyb30). Furthermore, along with the generation of two novel mutants with high yield and cold tolerance, this study also demonstrated CRISPR use in the simultaneous improvement of multiple agronomic traits.
To Sum up, the CRISPR/Cas can be used for site-specific genome editing in various crops to increase the productivity conferring biotic and abiotic stress resistance. The main advantage of using CRISPR is that multiple gene edits or Polymorphic QTL gene editing can be performed simultaneously in a single generation. Apart from this, its wide application in all sorts of plants and animals and flexibility to use in-vivo and in-vitro magnifies CRISPR’s popularity. With such a vast application area, it is pretty clear that it can play a crucial role in solving the food scarcity problem and contribute to survival.
Reference
Nekrasov V, Wang C, Win J, Lanz C, Weigel D, Kamoun S. Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion OPEN. Sci Rep. 2017;7. doi:10.1038/s41598-017- 00578-x
Zhang Y, Bai Y, Wu G, Zou S, Chen Y, Gao C, Tang D. Simultaneous modification of three homoeologs of TaEDR1 by genome editing enhances powdery mildew resistance in wheat. Plant J. 2017;91(4):714–24. doi:10.1111/tpj.13599
Zeng, Y., Wen, J., Zhao, W., Wang, Q., and Huang, W. (2020). Rational improvement of rice yield and cold tolerance by editing the three genes OsPIN5b, GS3, and OsMYB30 with the CRISPR-Cas9 System. Front. Plant Sci. 10:1663. doi: 10.3389/fpls.2019.01663
Zsögön, A., Čermák, T., Naves, E. R., Notini, M. M., Edel, K. H., Weinl, S., Freschi, L., Voytas, D. F., Kudla, J., & Peres, L. E. P. (2018). De novo domestication of wild tomato using genome editing. Nature Biotechnology, 36(12), 1211–1216. https://doi.org/10.1038/nbt.4272