Agriculture and climate change are internally correlated with each other in various aspects, as climate change is the main cause of biotic and abiotic stresses, which have adverse effects on the agriculture of a region. The land and its agriculture are being affected by climate changes in different ways, e.g., variations in annual rainfall, average temperature, heat waves, modifications in weeds, pests or microbes, global change of atmospheric CO2 or ozone level, and fluctuations in sea level. The threat of varying global climate has greatly driven the attention of scientists, as these variations are imparting negative impact on global crop production and compromising food security worldwide. According to some predicted reports, agriculture is considered the most endangered activity adversely affected by climate changes. To date, food security and ecosystem resilience are the most concerning subjects worldwide. Climate-smart agriculture is the only way to lower the negative impact of climate variations on crop adaptation, before it might affect global crop production drastically. In this review paper, we summarize the causes of climate change, stresses produced due to climate change, impacts on crops, modern breeding technologies, and biotechnological strategies to cope with climate change, in order to develop climate resilient crops. Revolutions in genetic engineering techniques can also aid in overcoming food security issues against extreme environmental conditions, by producing transgenic plants.

Impact of Climate Change on Crops Adaptation and Strategies to Tackle Its Outcome: A Review.

The availability of genome sequences for several crops and advances in genome editing approaches has opened up possibilities to breed for almost any given desirable trait. Advancements in genome editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) has made it possible for molecular biologists to more precisely target any gene of interest. However, these methodologies are expensive and time-consuming as they involve complicated steps that require protein engineering. Unlike first-generation genome editing tools, CRISPR/Cas9 genome editing involves simple designing and cloning methods, with the same Cas9 being potentially available for use with different guide RNAs targeting multiple sites in the genome. After proof-of-concept demonstrations in crop plants involving the primary CRISPR-Cas9 module, several modified Cas9 cassettes have been utilized in crop plants for improving target specificity and reducing off-target cleavage (eg. Nmcas9, Sacas9, Stcas9). Further, the availability of Cas9 enzymes from additional bacterial species has made available options to enhance specificity and efficiency of gene editing methodologies. This review summarizes the options available to plant biotechnologists to bring about crop improvement using CRISPR/Cas9 based genome editing tools and also presents studies where CRISPR/Cas9 has been used for enhancing biotic and abiotic stress tolerance. Application of these techniques will result in the development of non-genetically modified (Non-GMO) crops with the desired trait that can contribute to increased yield potential under biotic and abiotic stress conditions.

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CRISPR for Crop Improvement: An Update Review.

Rhizosphere microbiome: Engineering bacterial competitiveness for enhancing crop production.

Increasing agricultural productivity via modern breeding strategies is of prime interest to attain global food security. An array of biotic and abiotic stressors affect productivity as well as the quality of crop plants, and it is a primary need to develop crops with improved adaptability, high productivity, and resilience against these biotic/abiotic stressors. Conventional approaches to genetic engineering involve tedious procedures. State-of-the-art OMICS approaches reinforced with next-generation sequencing and the latest developments in genome editing tools have paved the way for targeted mutagenesis, opening new horizons for precise genome engineering. Various genome editing tools such as transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and meganucleases (MNs) have enabled plant scientists to manipulate desired genes in crop plants. However, these approaches are expensive and laborious involving complex procedures for successful editing. Conversely, CRISPR/Cas9 is an entrancing, easy-to-design, cost-effective, and versatile tool for precise and efficient plant genome editing. In recent years, the CRISPR/Cas9 system has emerged as a powerful tool for targeted mutagenesis, including single base substitution, multiplex gene editing, gene knockouts, and regulation of gene transcription in plants. Thus, CRISPR/Cas9-based genome editing has demonstrated great potential for crop improvement but regulation of genome-edited crops is still in its infancy. Here, we extensively reviewed the availability of CRISPR/Cas9 genome editing tools for plant biotechnologists to target desired genes and its vast applications in crop breeding research.

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Modern Trends in Plant Genome Editing: An Inclusive Review of the CRISPR/Cas9 Toolbox

Abiotic stresses such as extreme heat, cold, drought, and salt have brought alteration in plant growth and development, threatening crop yield and quality leading to global food insecurity. Many factors plays crucial role in regulating various plant growth and developmental processes during abiotic stresses. Ethylene response factors (ERFs) are AP2/ERF superfamily proteins belonging to the largest family of transcription factors known to participate during multiple abiotic stress tolerance such as salt, drought, heat, and cold with well-conserved DNA-binding domain. Several extensive studies were conducted on many ERF family proteins in plant species through over-expression and transgenics. However, studies on ERF family proteins with negative regulatory functions are very few. In this review article, we have summarized the mechanism and role of recently studied AP2/ERF-type transcription factors in different abiotic stress responses. We have comprehensively discussed the application of advanced ground-breaking genome engineering tool, CRISPR/Cas9, to edit specific ERFs. We have also highlighted our on-going and published R&D efforts on multiplex CRISPR/Cas9 genome editing of negative regulatory genes for multiple abiotic stress responses in plant and crop models. The overall aim of this review is to highlight the importance of CRISPR/Cas9 and ERFs in developing sustainable multiple abiotic stress tolerance in crop plants.

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

Plant annexins are Ca2+-dependent phospholipid-binding proteins and exist as multigene families in plants. They are implicated in the regulation of plant development as well as protection from environmental stresses. In this study, the rice annexin gene OsAnn3 knockout was performed via the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated proteins) mediated genome editing. Thus, mutant plantlets were successfully obtained. We identified cold tolerance phenotype of T1 mutant lines from T0 biallelic mutants using the 4∼6°C for 3 days cold treatment. The results showed that REC (the relative electrical conductivity) of T1 mutant lines was increased, and the survival ratio of T1 mutant lines was decreased dramatically compared with the wild type after the exposure to cold treatment. It was suggested that OsAnn3 was involved in cold tolerance of rice.

Knock out of the annexin gene OsAnn3 via CRISPR/Cas9-mediated genome editing decreased cold tolerance in rice

Abstract In prokaryotes, few studies have applied the flippase (FLP)/P1-flippase recombination target (LoxP-FRT) recombination system to switch gene expression. This study developed a new method for switching gene expression by constructing an FLP/LoxP-FRT site-specific recombination system in Escherichia coli. To this end, we placed the Nos terminator flanked by a pair of LoxP-FRT in front of enhanced green fluorescent protein (eGFP). The Nos terminator was used to block the expression of the eGFP. When a plasmid expressing FLP was available, deletion of the Nos terminator would allow expression of eGFP. The regulatory effect was demonstrated by eGFP expression. The efficiency of the gene switch was calculated as high as 89.67%. The results showed that the FLP/LoxP-FRT recombinase system could be used as a gene switch to regulate gene expression in prokaryotes. This new method for switching gene expression could simplify the gene function analysis in E. coli and other prokaryotes, as well as eukaryotes. Graphical abstract

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Application of the FLP/LoxP-FRT recombination system to switch the eGFP expression in a model prokaryote

Climate change has caused high salinity in many fields, particularly in the mud flats in coastal regions. The resulting salinity has become one of the most significant abiotic stresses affecting the world’s rice crop productivity. Developing elite cultivars with novel salinity-tolerance traits is regarded as the most cost-effective and environmentally friendly approach for utilizing saline-alkali land. To develop a highly efficient green strategy and create novel rice germplasms for salt-tolerant rice breeding, this study aimed to improve rice salinity tolerance by combining targeted CRISPR/Cas9-mediated editing of the OsRR22 gene with heterosis utilization. The novel alleles of the genic male-sterility (GMS) and elite restorer line (733Srr22-T1447-1 and HZrr22-T1349-3) produced 110 and 1 bp deletions at the third exon of OsRR22 and conferred a high level of salinity tolerance. Homozygous transgene-free progeny were identified via segregation in the T2 generation, with osrr22 showing similar agronomic performance to wild-type (733S and HZ). Furthermore, these two osrr22 lines were used to develop a new promising third-generation hybrid rice line with novel salinity tolerance. Overall, the results demonstrate that combining CRISPR/Cas9 targeted gene editing with the “third-generation hybrid rice system” approach allows for the efficient development of novel hybrid rice varieties that exhibit a high level of salinity tolerance, thereby ensuring improved cultivar stability and enhanced rice productivity.

Novel Salinity-Tolerant Third-Generation Hybrid Rice Developed via CRISPR/Cas9-Mediated Gene Editing

Apomixis can fix the heterosis of Hybrid F1, by maintaining its heterozygous genotype, and is an ideal way for the development of hybrid rice. In this paper, we designed an engineering strategy for realizing apomictic reproduction of hybrid rice in the way of induce adventitious embryos. An embryogenesis gene, AtWUS, controlled by the ovule-specific promoter, a ribonuclease gene Barnase driven by the egg cell-specific promoter pDD45, and an inactivation gene ZmAA1 driven by the pollen-specific promoter pG47 were simultaneously integrated into one T-DNA, and co-transformed with the second T-DNA carrying a Barstar gene. Double-seedlings were observed in transgenic line. Whole-genome sequencing and ploidy levels confirmed by flow cytometry showed that one of the double-seedlings was heterozygous diploid and the other seedling was homozygous haploid, which confirmed that embryogenesis in one of the double-seedlings arises from the zygote after fertilization and the other derived from an unfertilized gamete. Meanwhile we obtained embryo-free seeds at frequencies of 2.6% to 3.8% in T1 generation, and 0.75% to 3% in T2 generation. Though we did not obtained adventitious embryos in hybrid rice in this study, the phenomenon of double-seedlings and embryo-free seeds in transgenic line was informative and strongly suggested that endosperm development is an autonomously organized process in rice, independent of egg cell fertilization and embryo-endosperm communication. This provides novel insights into the induction of haploid embryos and lends theoretical support to successful clonal propagation using synthetic apomixis

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Ning Tang

Papers

Knock out of the annexin gene OsAnn3 via CRISPR/Cas9-mediated genome editing decreased cold tolerance in rice

Application of the FLP/LoxP-FRT recombination system to switch the eGFP expression in a model prokaryote

Novel Salinity-Tolerant Third-Generation Hybrid Rice Developed via CRISPR/Cas9-Mediated Gene Editing

Double-seedlings and embryo-free seeds generated by genetic engineering