Wheat (Triticum aestivum L.) is a staple food crop consumed by more than 30% of world population. Nitrogen (N) fertilizer has been applied broadly in agriculture practice to improve wheat yield to meet the growing demands for food production. However, undue N fertilizer application and the low N use efficiency (NUE) of modern wheat varieties are aggravating environmental pollution and ecological deterioration. Under nitrogen-limiting conditions, the rice (Oryza sativa) abnormal cytokinin response1 repressor1 (are1) mutant exhibits increased NUE, delayed senescence and consequently, increased grain yield. However, the function of ARE1 ortholog in wheat remains unknown. Here, we isolated and characterized three TaARE1 homoeologs from the elite Chinese winter wheat cultivar ZhengMai 7698. We then used CRISPR/Cas9-mediated targeted mutagenesis to generate a series of transgene-free mutant lines either with partial or triple-null taare1 alleles. All transgene-free mutant lines showed enhanced tolerance to N starvation, and showed delayed senescence and increased grain yield in field conditions. In particular, the AABBdd and aabbDD mutant lines exhibited delayed senescence and significantly increased grain yield without growth defects compared to the wild-type control. Together, our results underscore the potential to manipulate ARE1 orthologs through gene editing for breeding of high-yield wheat as well as other cereal crops with improved NUE.

Increasing yield potential through manipulating of an ARE1 ortholog related to nitrogen use efficiency in wheat by CRISPR/Cas9

Beneficial alleles derived from local landraces or related species, or even orthologs from other plant species, are often caused by differences of one or several single-nucleotide polymorphisms or indels in either the promoter region or the encoding region of a gene and often account for major differences in agriculturally important traits. Clustered regularly interspaced short palindromic repeats-associated endonuclease Cas9 system (CRISPR/Cas9)-mediated precision genome editing enables targeted allele replacement or insertion of flag or foreign genes at specific loci via homology-directed repair (HDR); however, HDR efficiency is low due to the intrinsic rare occurrence of HDR and insufficient DNA repair template in the proximity of a double-stranded break (DSB). Precise replacement of the targeted gene with elite alleles from landraces or relatives into a commercial variety through genome editing has been a holy grail in the crop genome editing field. In this update, we briefly summarize CRISPR/Cas-mediated HDR in plants. We describe diverse strategies to improve HDR efficiency by manipulating the DNA repair pathway, timing DSB induction, and donor delivery, and so on. Lastly, we outline open questions and challenges in HDR-mediated precision genome editing in both plant biological research and crop improvement. 

/pdf/oup-accepted-manuscript-95jkbhkm.pdf

OUP accepted manuscript

Beneficial alleles derived from local landraces or related species, or even orthologs from other plant species, are often caused by differences of one or several single nucleotide polymorphisms (SNPs) or indels in either the promoter region or the encoding region of a gene and often account for major differences in agriculturally important traits. CRISPR/Cas9-mediated precision genome editing enables targeted allele replacement or insertion of flag or foreign genes at specific loci via homology-directed repair (HDR); however, HDR efficiency is low due to the intrinsic rare occurrence of HDR and insufficient DNA repair template (DRT) in the proximity of a double-stranded break (DSB). Precise replacement of the targeted gene with elite alleles from landraces or relatives into a commercial variety through genome editing has been a holy grail in the crop genome editing field. In this update, we briefly summarize CRISPR/Cas-mediated HDR in plants. We describe diverse strategies to improve HDR efficiency by manipulating the DNA repair pathway, timing DSB induction and donor delivery, etc. Lastly, we outline open questions and challenges in HDR-mediated precision genome editing in both plant biological research and crop improvement.

/pdf/an-update-on-precision-genome-editing-by-homology-directed-2kdws4il.pdf

An update on precision genome editing by homology-directed repair in plants.

CRISPR/Cas technology has recently become the molecular tool of choice for gene function studies in plants as well as crop improvement. Wheat is a globally important staple crop with a well annotated genome and there is plenty of scope for improving its agriculturally important traits using genome editing technologies, such as CRISPR/Cas. As part of this study we targeted three different genes in hexaploid wheat Triticum aestivum: TaBAK1-2 in the spring cultivar Cadenza as well as Ta-eIF4E and Ta-eIF(iso)4E in winter cultivars Cezanne, Goncourt and Prevert. Primary transgenic lines carrying CRISPR/Cas-induced indels were successfully generated for all targeted genes. While BAK1 is an important regulator of plant immunity and development, Ta-eIF4E and Ta-eIF(iso)4E act as susceptibility (S) factors required for plant viruses from the Potyviridae family to complete their life cycle. We anticipate the resultant homozygous tabak1-2 mutant lines will facilitate studies on the involvement of BAK1 in immune responses in wheat, while ta-eif4e and ta-eif(iso)4e mutant lines have the potential to become a source of resistance to wheat spindle streak mosaic virus (WSSMV) and wheat yellow mosaic virus (WYMV), both of which are important pathogens of wheat. As winter wheat varieties are generally less amenable to genetic transformation, the successful experimental methodology for transformation and genome editing in winter wheat presented in this study will be of interest to the research community working with this crop.

/pdf/efficient-crispr-cas-mediated-targeted-mutagenesis-in-spring-nco6azfz3l.pdf

Efficient CRISPR/Cas-Mediated Targeted Mutagenesis in Spring and Winter Wheat Varieties.

Alleviating micronutrients associated problems in children below five years and women of childbearing age, remains a significant challenge, especially in resource-poor nations. One of the most important staple food crops, wheat attracts the highest global research priority for micronutrient (Fe, Zn, Se, and Ca) biofortification. Wild relatives and cultivated species of wheat possess significant natural genetic variability for these micronutrients, which has successfully been utilized for breeding micronutrient dense wheat varieties. This has enabled the release of 40 biofortified wheat cultivars for commercial cultivation in different countries, including India, Bangladesh, Pakistan, Bolivia, Mexico and Nepal. In this review, we have systematically analyzed the current understanding of availability and utilization of natural genetic variations for grain micronutrients among cultivated and wild relatives, QTLs/genes and different genomic regions regulating the accumulation of micronutrients, and the status of micronutrient biofortified wheat varieties released for commercial cultivation across the globe. In addition, we have also discussed the potential implications of emerging technologies such as genome editing to improve the micronutrient content and their bioavailability in wheat.

/pdf/wheat-biofortification-utilizing-natural-genetic-diversity-30jf77uc.pdf

Wheat Biofortification: Utilizing Natural Genetic Diversity, Genome-Wide Association Mapping, Genomic Selection, and Genome Editing Technologies

Wheat (Triticum aestivum, 2n = 6x = 42, AABBDD) is one of the most important staple food crops in the world. Despite the fact that wheat production has significantly increased over the past decades, future wheat production will face unprecedented challenges from global climate change, increasing world population, and water shortages in arid and semi-arid lands. Furthermore, excessive applications of diverse fertilizers and pesticides are exacerbating environmental pollution and ecological deterioration. To ensure global food and ecosystem security, it is essential to enhance the resilience of wheat production while minimizing environmental pollution through the use of cutting-edge technologies. However, the hexaploid genome and gene redundancy complicate advances in genetic research and precision gene modifications for wheat improvement, thus impeding the breeding of elite wheat cultivars. In this review, we first introduce state-of-the-art genome-editing technologies in crop plants, especially wheat, for both functional genomics and genetic improvement. We then outline applications of other technologies, such as GWAS, high-throughput genotyping and phenotyping, speed breeding, and synthetic biology, in wheat. Finally, we discuss existing challenges in wheat genome editing and future prospects for precision gene modifications using advanced genome-editing technologies. We conclude that the combination of genome editing and other molecular breeding strategies will greatly facilitate genetic improvement of wheat for sustainable global production.

Chen Zhang

Papers

Present and future prospects for wheat improvement through genome editing and advanced technologies.