Evolvement of transgenic male-sterility and fertility-restoration system in rice for production of hybrid varieties.

KEY MESSAGE: We have developed a unique male-sterility and fertility-restoration system in rice by combining Brassica napus cysteine-protease gene (BnCysP1) with anther-specific P12 promoter of rice for facilitating production of hybrid varieties. In diverse crop plants, male-sterility has been exploited as a useful approach for production of hybrid varieties to harness the benefits of hybrid vigour. The promoter region of Os12bglu38 gene of rice has been isolated from the developing panicles and was designated as P12. The promoter was fused with gusA reporter gene and was expressed in Arabidopsis and rice systems. Transgenic plants exhibited GUS activity in tapetal cells and pollen of the developing anthers indicating anther/pollen-specific expression of the promoter. For engineering nuclear male sterility, the coding region of Brassica napus cysteine protease1 (BnCysP1) was isolated from developing seeds and fused to P12 promoter. Transgenic rice plants obtained with P12-BnCysP1 failed to produce functional pollen grains. The F1 seeds obtained from BnCysP1 male-sterile plants and untransformed controls showed 1:1 (tolerant:sensitive) ratio when germinated on the MS medium supplemented with phosphinothricin (5 mg/l), confirming that the male sterility has been successfully engineered in rice. For male fertility restoration, transgenic rice plants carrying BnCysP1Si silencing system were developed. The pollination of BnCysP1 male-sterile (female-fertile) plants with BnCysP1Si pollen resulted in normal grain filling. The F1 seeds of BnCysP1 × BnCysP1Si when germinated on the MS basal medium containing PPT (5 mg/l) and hygromycin (70 mg/l) exhibited 1:1 (tolerant:sensitive) ratio and the tolerant plants invariably showed normal grain filling. The overall results clearly suggest that the customized male-sterility & fertility-restoration system can be exploited for quality hybrid seed production in various crops.

Synthetic directed evolution in plants: unlocking trait engineering and improvement.

Genetic variation accelerates adaptation and resilience and enables the survival of species in their changing environment. Increasing the genetic diversity of crop species is essential to improve their yield and enhance food security. Synthetic directed evolution (SDE) employs localized sequence diversification (LSD) of gene sequence and selection pressure to evolve gene variants with better fitness, improved properties and desired phenotypes. Recently, CRISPR-Cas-dependent and -independent technologies have been applied for LSD to mediate synthetic evolution in diverse species, including plants. SDE holds excellent promise to discover, accelerate and expand the range of traits of the value in crop species. Here, we highlight the efficient SDE approaches for the LSD of plant genes, selection strategies and critical traits for targeted improvement. We discuss the potential of emerging technologies, including CRISPR-Cas base editing, retron editing, EvolvR and prime editing, to establish efficient SDE in plants. Moreover, we cover CRISPR-Cas-independent technologies, including T7 polymerase editor for continuous evolution. We highlight the key challenges and potential solutions of applying SDE technologies to improve the plant traits of the value.

CRISPR-Based Crop Improvements: A Way Forward to Achieve Zero Hunger.

Zero hunger is one of the sustainable development goals set by the United Nations in 2015 to achieve global food security by 2030. The current harvest of crops is insufficient; feeding the world's population and meeting the goal of zero hunger by 2030 will require larger and more consistent crop production. Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR-Cas) technology is widely used for the plant genome editing. In this review, we consider this technology as a potential tool for achieving zero hunger. We provide a comprehensive overview of CRISPR-Cas technology and its most important applications for food crops' improvement. We also conferred current and potential technological breakthroughs that will help in breeding future crops to end global hunger. The regulatory aspects of deploying this technology in commercial sectors, bioethics, and the production of transgene-free plants are also discussed. We hope that the CRISPR-Cas system will accelerate the breeding of improved crop cultivars compared with conventional breeding and pave the way toward the zero hunger goal.

Genome Editing Technologies for Rice Improvement: Progress, Prospects, and Safety Concerns.

Rice (Oryza sativa) is an important staple food crop worldwide; to meet the growing nutritional requirements of the increasing population in the face of climate change, qualitative and quantitative traits of rice need to be improved. Stress-tolerant crop varieties must be developed with stable or higher yields under stress conditions. Genome editing and speed breeding have improved the accuracy and pace of rice breeding. New breeding technologies including genome editing have been established in rice, expanding the potential for crop improvement. Recently, other genome editing techniques such as CRISPR-directed evolution, CRISPR-Cas12a, and base editors have also been used for efficient genome editing in rice. Since rice is an excellent model system for functional studies due to its small genome and close syntenic relationships with other cereal crops, new genome-editing technologies continue to be developed for use in rice. In this review, we focus on genome-editing tools for rice improvement to address current challenges and provide examples of genome editing in rice. We also shed light on expanding the scope of genome editing and systems for delivering homology-directed repair templates. Finally, we discuss safety concerns and methods for obtaining transgene-free crops.

iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2.

The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19.

Development of an inducible male-sterility system in rice through pollen-specific expression of l-ornithinase (argE) gene of E. coli.

In the present investigation, an inducible male-sterility system has been developed in the rice. In order to introduce inducible male-sterility, the coding region of l-ornithinase (argE) gene of E. coli was fused to the Oryza sativa indica pollen allergen (OSIPA) promoter sequence which is known to function specifically in the pollen grains. Transgenic plants were obtained with argE gene and the transgenic status of plants was confirmed by PCR and Southern blot analyses. RT-PCR analysis confirmed the tissue-specific expression of argE in the anthers of transgenic rice plants. Transgenic rice plants expressing argE, after application of N-acetyl-phosphinothricin (N-ac-PPT), became completely male-sterile owing to the pollen-specific expression of argE. However, argE-transgenic plants were found to be self fertile when N-ac-PPT was not applied. Normal fertile seeds were obtained from the cross pollination between male-sterile argE transgenics and untransformed control plants, indicating that the female fertility is not affected by the N-ac-PPT treatment. These results clearly suggest that the expression of argE gene affects only the male gametophyte but not the gynoecium development. Induction of complete male-sterility in the rice is a first of its kind, and moreover this male- sterility system does not require the deployment of any specific restorer line.