CRISPR-Cas9 Directed Genome Modification for Abiotic Stress Tolerance Rice

CRISPR Clustered regularly interspaced short palindromic repeats. CRISPR-associated protein (Cas) technology for site-specific genome editing has been used to precisely induce mutagenesis in a variety of plant species, including rice. Because Cas9 causes blunt-ended double-strand breaks, which are then repaired without substantial end-processing, a high fraction of mutations created by CRISPR/Cas9 is extremely short insertions and deletions. CRISPR/Cas9 is a powerful tool for targeted mutagenesis in rice, as well as some essential crops. One of the most demanding environmental restrictions affecting agricultural output around the world is salinity and drought. Because plant adaptation to abiotic stress is polygenic, much more rice genes have that play a critical role in abiotic stress response

The CRISPR-Cas system, the most recent genome editing approach, evolved from the adaptive immune system of bacteria and archaea, which allows organisms to adaptive immunity against the bacteriophage virus. CRISPR- clustered regularly interspaced short palindromic repeat was discovered- 2002 [1-3]. Though all CRISPR-Cas systems have DNA repeats, spacers, and Cas genes in common, the system has a great deal of variability due to fast evolution and horizontal gene transfer in nature. The CRISPR-Cas systems were classified using a multi-criteria approach based on characteristic Cas genes (Cas1, Cas2) and the sequence is very much similar to Cas proteins, Cas1, and also the structural arrangement of the system in the loci. In the genome-editing technique, accuracy in base editing has always been a challenge [4-8]. By combining catalytically inactive Cas9 variants, dCas9 (dead Cas9), and Cas9 nickase to target deaminase domains and edit specific loci, efforts are being undertaken to improve the accuracy of gene editing schemes. Other than the CRISPR/Cas9 system, recent findings have revealed a variety of possible tools, including type V systems having the DNA-targeting Cas12 (Cpf1 or C2c1) effectors and RNA-targeting type VI systems containing the Cas13 (C2c2).

Rice genome editing with Cas9 system

All available internet tools were used to design and synthesize sgRNA, starting with the selection of the target gene. The sgRNA has been cloned into a plant binary vector along with the necessary Cas9 or Cas12 variants for transformation into target plant species using an acceptable approach such as Agrobacterium-mediated transformation. The presence of Cas9 or Cas12 and sgRNA would be tested after transformation on the putatively transformed plants. The plants are then screened for the desired targeted mutations using PCR-RE genotyping and DNA sequencing techniques, followed by the creation of transgenic seeds [9-16].

The Applications of CRISPR/Cas9 technology in plant science

In rice Gene research is a field of study that looks into how genes work. There are Abiotic stress (salt, drought, cold, heat, etc.) and biotic stress are (bacteria, viruses, fungi, etc.) conditions that are posing hazards to agriculture around the world [17-25]. breeders are always pursuing yield gains, quality improvement, and stress tolerance/ resistance as a result of the global population increase, shortages of food, and degradation of the environment. Genome editing through CRISPR/Cas9-technology can be used for more than only functional genomics areas; it can also be used to improve crop types. CRISPR/Cas9 has the potential to be extremely useful in many aspects of plant breeding, both today and in the future.

The major challenges for CRISPR/Cas9 in Crop

CRISPR/Cas9 gene-editing system has a wide range of possible uses in plant science. The narrow pool of genes influencing essential agronomic features, which are a requirement for employing this technology, is a big barrier. In this regard, deciphering genomic sequencing data and targeting the genetic data for crop development are urgently needed. Other obstacles include inefficient transformation systems and difficulty with plant tissue cultures and regeneration, both of which necessitate an intricate, tedious, and time-taking process. The possibility for off-target consequences, as well as the safety concerns associated with CRISPR regenerated bioproducts, is currently being debated. Off-target effects are, fortunately, more acceptable in plants than in animals, particularly humans. Plant mutants having off-target effects can be found and deleted via segregationduring successive crosses as detection technologies improve. Designing appropriate sgRNAs with a strong affinity for targeted reason sequences and selecting a Cas9 nuclease with high processivity, in combination with the proper experimental procedure, may help to overcome off-target effects in the future. The lack of market access for genome-edited crops is another important issue [26-30].

CRISPR/Cas9 technologies have opened up new possibilities in the domain of plant genetic manipulation. CRISPR has several desirable qualities for a proper gene editing system for site-specific mutagenesis, high specificity, high processivity, cheap cost, high efficiency, and very simple perform in the lab, which is not possible with old mutation techniques. The CRISPR system is also highly developed to the ZFN and TALEN gene-editing system because the Cas9 nuclease is guided by RNA rather than proteins. Because the CRISPR/Cas9 system is reasonably well known, its construct methods have been developed now to this day, and efforts to limit off-target effects have been made. Special toolkits for the easy creation of CRISPR/Cas9 systems are also being developed, the development approaches for CRISPR/Cas9 created cut screening it can also use a single transformation event to modify a single gene or a greater number of genes. Furthermore. All of these benefits have prompted the researcher’s use of CRISPR/Cas9 in crop improvement and plant molecular research, particularly for traits related to high yield quality, biotic stress, and abiotic stress resistance. CRISPR/Cas9 is also appealing because it has the ability to produce gene-edited crops. As a result, CRISPR technology appears to be the best tool for plant molecular research area.

1. Abdallah MMS, Abdelgawad ZA, El-Bassiouny HMS. Alleviation of the adverse effects of salinity stress using trehalose in two rice varieties. South African J Botany. 2016; 103: 275-282.
2. Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V. Editing plant genomes with CRISPR/Cas9. Curr Opin Biotech. 2015; 32: 76-84.
3. Bo W, Zhaohui Z, Huanhuan Z, Xia W, Binglin L, Lijia Y, et al. Targeted mutagenesis of NAC transcription factor gene, OsNAC041, leading to salt sensitivity in rice. Rice Sci. 2019; 26: 98-108.
4. Borrelli VM, Brambilla V, Rogowsky P, Marocco A, Lanubile A. The enhancement of plant disease resistance using CRISPR/Cas9 technology. Frontiers Plant Sci. 2018.
5. Chen G, Hu J, Dong L, Zeng D, Guo L, Zhang G, et al. The tolerance of salinity in rice requires the presence of a functional copy of FLN2. Bio. 2020; 10: 17.
6. Debbarma J, Sarki YN, Saikia B, Boruah HPD, Singha D, Chikkaputtaiah C. Ethylene response factor (ERF) family proteins in abiotic stresses and CRISPR–Cas9 genome editing of ERFs for multiple abiotic stress tolerance in crop plants: a review. Molecular bio. 2019; 61: 153-172.
7. Endo M, Mikami M, Toki S. Multigene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice. Plant Cell Phi. 2015; 56: 41-47.
8. Ganie SA, Wani SH, Henry R, Hensel G. Improving rice salt tolerance by precision breeding in a new era. Curr Opinion Plant Bio. 2021.
9. Liang-Fa G. Overexpression of the trehalose-6-phosphate phosphatase gene OsTPP1 confers stress tolerance in rice and results in the activation of stress responsive genes. Planta. 2008; 228: 191-201.
10. Islam OM. Functional identification of a rice trehalase gene involved in salt stress tolerance. Gene. 2019; 685: 42-49.
11. Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res. 2013; 41: 188.
12. Khan I, Khan S, Zhang Y, Zhou J, Akhoundian M. CRISPR-Cas technology based genome editing for modification of salinity stress tolerance responses in rice (Oryza sativa L.). Mol Biol Rep. 2021; 48: 3605-3615.
13. Ge L, Chao D, Shi M, Zhu M, Gao J, Lin H. Overexpression of the trehalose-6-phosphate synthase gene OsTPS1 enhances abiotic stress tolerance in rice. Planta. 2011; 234: 1007-1018.
14. Li J, Sun Y, Du J, Zhao Y, Xia L. Generation of targeted point mutations in rice by a modified CRISPR/Cas9 system. Molecular Plant. 2017; 10: 526-529.
15. Liu X, Wu S, Xu J, Sui C, Wei J. Application of CRISPR/Cas9 in plant biology. Acta pharmaceutica sinica B. 2017; 7: 292-302.
16. Nounjan N, Nghia TP, Theerakulpisut P. Exogenous proline and trehalose promote recovery of rice seedlings from salt-stress and differentially modulate antioxidant enzymes and expression of related genes. J Plant Phi. 2012; 6: 596-604.
17. Paul J, Qi Y. CRISPR/Cas9 for plant genome editing: accomplishments, problems and prospects. Plant Cell Rep. 2016; 35: 1417-1427.
18. Mark CFR. Accumulation of trehalose increases soluble sugar contents in rice plants conferring tolerance to drought and salt stress. Plant Bio Reports. 2012; 6: 89-96.
19. Samantaray MUS, Sahu S. Characterization and cellulase production activity of different aspergillus sp. isolated from “puri, odisha” marine fungal strains. Int J Res Applied Sci Bio. 2021; 8: 71-87.
20. Samantaray MUS, Mishra MA, Singh YD. Biosynthesis and antimicrobial activities of silver nanoparticles (agnps) by using leaf extracts of tagetes erecta (marigold) and tridax procumbens (Tridax). Int J Res Applied Sci Bio; 2020; 7: 209-226.
21. Santosh Kumar VV, Verma RK, Yadav SK, Yadav P, Watts A, Rao M V, et al. CRISPR-Cas9 mediated genome editing of drought and salt tolerance (OsDST) gene in indica mega rice cultivar MTU1010. Phi Molecular Bio Plants. 2020; 26: 1099-1110.
22. Shen C, Que Z, Xia Y, Tang N, Li D, He R. Knock out of the annexin gene OsAnn3 via CRISPR/Cas9-mediated genome editing decreased cold tolerance in rice. J Plant Bio. 2017; 60: 539-547.
23. Tang L, Mao B, Li Y, Lv Q, Zhang L, Chen C, et al. Knockout of OsNramp5 using the CRISPR/Cas9 system produces low Cd-accumulating indica rice without compromising yield. Scientific Rep. 2017; 7: 1-12.
24. Vishal B. Os TPS 8 controls yield?related traits and confers salt stress tolerance in rice by enhancing suberin deposition. New Phytologist. 2019; 221: 1369-1386.
25. Wang F, Wang C, Liu P, Lei C, Hao W, Gao Y, et al. Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922. PloS one. 2016.
26. Wang X, He Y, Wei H, Wang L. A clock regulatory module is required for salt tolerance and control of heading date in rice. Plant Cell Environment. 2021; 44: 3283-3301.
27. Zhang A, Liu Y, Wang F, Li T, Chen Z, Kong D. Enhanced rice salinity tolerance via CRISPR/Cas9-targeted mutagenesis of the OsRR22 gene. Molecular Breeding. 2019; 39: 1-10.
28. Zhou H, Liu B, Weeks DP, Spalding MH, Yang B. Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Res. 2014.
29. Zhou J, Deng K, Cheng Y, Zhong Z, Tian L, Tang X, et al. CRISPR-Cas9 based genome editing reveals new insights into microRNA function and regulation in rice. Frontiers Plant Sci. 2017; 8.
30. Zhou J, Yuan M, Zhao Y, Quan Q, Yu D, Yang, H, et al. Efficient deletion of multiple circle RNA loci by CRISPR?Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice. Plant Biotechnol J. 2021; 19: 1240-1252.