Ever growing human population, decreasing arable land, scarcity of water, and changing climate have raised several concerns for global food production. Though conventional plant breeding contributed significantly to food production by developing high yielding varieties or hybrids, it has its own drawback with respect to turnaround time to develop new varieties and lack of germplasm source for novel traits. Introducing these traits (genes) from other genera through transgenic approach will attract many biosafety regulations to commercialize the products. However, new technology like CRISPR can be a solution to the many existing problems in agriculture production scenario. The gene-editing technology can be used to specifically alter the gene sequences of specific traits in a specific variety rather without shuffling the genetic constellation of a variety which is already being cultivated by the farming community. Initially, the type-II CRISPR technique was used to edit the target gene/s using guideRNA (gRNA) and Cas9 endonucleases that induce double-strand breaks repaired primarily by Non-Homologous End Joining (NHEJ) and trivially by Homologous Directed Repair (HDR)1.
The mutant alleles of the drought and salt tolerance (DST) gene were created using CRISPR-Cas9 technology in the indica rice variety MTU1010 using two guide RNAs. The DST gene was knocked out and the mutant dstΔ184-205 with 366 bp deletion was selected that exhibited moderate tolerance to osmotic stress and high salt tolerance2. Even though CRISPR-Cas is utilized for targeted editing its efficiency is hampered by off-targets and unintended mutations through NHEJ. To address these challenges, the variants of CRISPR-Cas viz., nCas9 (nickase Cas9), dCas9 (deadCas9), Cpf1, Cas12, and Cas13 were explored. Additionally, novel techniques like base editing and prime editing offer precision editing without double-stranded breaks or external repair templates. Despite this, base editing and prime editing face challenges like off-target effects, bystander mutations, and reduced efficiency.
To subdue this, a novel approach NICER (multiple Nicks induced by Cas9 Nickase and a homologous Chromosome as Endogenous Repair template) has been introduced. NICER aims to correct heterozygous mutations by creating multiple nicks in the target gene. The repair process involves utilizing the sister chromosome as a repair template through multiple nicks induced Inter homolog-homologous recombination (MN-IH-HR), offering a promising solution to enhance precision in genetic modifications3.
The evolving precision genome editing techniques like base editing, prime editing, and NICER are pivotal in transforming genetic modifications for crop enhancement. Base and prime editing techniques have been already encashed in many crops like rice, corn, wheat, tomato, cucumber, apple, etc. So, products developed from CRISPR gene-editing will be most promising to solve many burning issues in agriculture at present as well as in near future. The integration of these advanced technologies signals a new era of efficient and targeted strategies for enhancing crops performance. Varieties developed through these gene editing techniques may not require extensive yield testing experiments because the performance of the variety being considered for editing is well established.
References:
1 CHEN, K., WANG, Y., ZHANG, R., ZHANG, H. AND GAO, C., 2019, CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu. Rev. Plant Biol., 70: 667-697.
2 SANTOSH KUMAR, V.V., VERMA, R. K., YADAV, S. K., YADAV, P., WATTS, A., RAO, M. V. AND CHINNUSAMY, V., 2020, CRISPR-Cas9 mediated genome editing of drought and salt tolerance (OsDST) gene in indica mega rice cultivar MTU1010. Physiol. Mol. Biol. Plants, 26: 1099-1110.
3 TOMITA, A., SASANUMA, H., OWA, T., NAKAZAWA, Y., SHIMADA, M., FUKUOKA, T., OGI, T. AND NAKADA, S., 2023, Inducing multiple nicks promotes interhomolog homologous recombination to correct heterozygous mutations in somatic cells. Nat. Commun., 14(1): 5607.
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