CRISPR/Cas9 and Nanotechnology Pertinence in Agricultural Crop Refinement

Abstract

The CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) method is a versatile technique that can be applied in crop refinement. Currently, the main reasons for declining agricultural yield are global warming, low rainfall, biotic and abiotic stresses, in addition to soil fertility issues caused by the use of harmful chemicals as fertilizers/additives. The declining yields can lead to inadequate supply of nutritional food as per global demand. Grains and horticultural crops including fruits, vegetables, and ornamental plants are crucial in sustaining human life. Genomic editing using CRISPR/Cas9 and nanotechnology has numerous advantages in crop development. Improving crop production using transgenic-free CRISPR/Cas9 technology and produced fertilizers, pesticides, and boosters for plants by adopting nanotechnology-based protocols can essentially overcome the universal food scarcity. This review briefly gives an overview on the potential applications of CRISPR/Cas9 and nanotechnology-based methods in developing the cultivation of major agricultural crops. In addition, the limitations and major challenges of genome editing in grains, vegetables, and fruits have been discussed in detail by emphasizing its applications in crop refinement strategy.

Keywords: Cas9; Cas9 activators; biotic and abiotic stress; horticultural crops; nano-fertilizers; nanoparticles; nutritional value.

FIGURE 1

Targeted genome editing via CRISPR-Cas9. (A) The CRISPR-Cas9 system comprises of a Cas9 protein and guide RNA. Guide RNAs regulate the target DNA specificity by sequence complementarity. (B) gRNA and Cas9 protein form a binary complex that specifically cleaves target DNA creating a double-strand DNA break. (C) Cellular DNA repair mechanisms: non-homologous end joining (NHEJ) and homology-directed repair (HDR), repairs the double strand DNA break. In the process, short insertions, deletions, nucleotide substitutions, or gene insertion may occur. Reproduced with permission from El-Mounadi et al. (2020) Frontiers.

FIGURE 2

Schematic illustration of the steps involved in CRISPR/Cas9 Genetic Transformation; (A) Specific gene selection, (B) sgRNA designing for the specific gene, (C) vector construction, (D) transformation of the CRISPR/Cas9 system via Agrobacterium, (E) callus induction from agrobacterium infected explants, (F) plant regeneration from callus, (G) T₀ CRISPR/Cas9 mutated transgenic plants, (H) screening of transgenic plants by PCR, (I) identification of mutated plants by T7E1, (J) detection of transgenics by sanger sequencing, (K) various techniques to detect transgenic plants, (L) self-pollination of T₀ transgenic plants for generation of homozygous T1 plants, (M) mutated T₀ seeds, (N) Generation of transgene- free T₁ progeny, (O) Phenotypic analysis of T₁ plants. Reproduced with permission from Manghwar et al. (2019) CellPress.

FIGURE 3

Nanotechnology-based agriculturally important nano-fertilizers, which are increasing the agronomic productivity, efficiency, and reduce environmental stress. Although showed the improved applications in agriculture by nanotechnology and Types of stresses overcome by nanotechnology. Reproduced with permission from Mittal et al. (2020) Frontiers.

FIGURE 4

Nanomaterial-mediated plant genetic engineering. Functionalized nanomaterials can provide a delivery platform that is capable of traversing barriers (e.g., multilayered cell walls) to deliver exogenous plasmid DNA (pDNA) and siRNA into intact plant cells. CNTs, carbon nanotubes; MSN, mesoporous silica nanoparticles; MNP, magnet nanoparticles. Reproduced with permission from Peng et al. (2019) CellPress.