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The potential of genome editing to improve the agronomic performance of crops is often limited by low plant regeneration efficiencies and few transformable genotypes. Here, we show that expression of a fusion protein combining wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) substantially increases the efficiency and speed of regeneration in wheat, triticale and rice and increases the number of transformable wheat genotypes. GRF4–GIF1 transgenic plants were fertile and without obvious developmental defects. Moreover, GRF4–GIF1 induced efficient wheat regeneration in the absence of exogenous cytokinins, which facilitates selection of transgenic plants without selectable markers. We also combined GRF4–GIF1 with CRISPR–Cas9 genome editing and generated 30 edited wheat plants with disruptions in the gene Q ( AP2L-A5 ). Finally, we show that a dicot GRF–GIF chimera improves regeneration efficiency in citrus, suggesting that this strategy can be applied to dicot crops. A method to increase plant regeneration efficiency extends gene editing to more species and genotypes.

Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized. Protocols for crop genome editing would ideally be quick, efficient and specific while avoiding integration of transgenes into the genome of edited plants. Here, Liang et al . show that CRISPR/Cas9 ribonucleoproteins can be used to generate genome edited wheat plants in as little as nine weeks.

Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research. Plant genome editing typically relies upon transgenic intermediates, which is a concern given the current regulatory requirements concerning GMOs. Here, Zhang et al . describe a method to edit wheat genomes by transiently expressing CRISPR/Cas9 DNA or RNA, and are able to generate mutant plants with no detectable transgenes.

The conversion of lignocellulosic feedstocks to fermentable sugar for biofuel production is inefficient, and most strategies to enhance efficiency directly target lignin biosynthesis, with associated negative growth impacts. Here we demonstrate, for both laboratory- and field-grown plants, that expression of Pag-miR408 in poplar ( Populus alba × P. glandulosa ) significantly enhances saccharification, with no requirement for acid-pretreatment, while promoting plant growth. The overexpression plants show increased accessibility of cell walls to cellulase and scaffoldin cellulose-binding modules. Conversely, Pag-miR408 loss-of-function poplar shows decreased cell wall accessibility. Overexpression of Pag-miR408 targets three Pag-LACCASES , delays lignification, and modestly reduces lignin content, S/G ratio and degree of lignin polymerization. Meanwhile, the LACCASE loss of function mutants exhibit significantly increased growth and cell wall accessibility in xylem. Our study shows how Pag-miR408 regulates lignification and secondary growth, and suggest an effective approach towards enhancing biomass yield and saccharification efficiency in a major bioenergy crop. Modifying plant lignin pathway to enhance saccharification efficiency is often associated with growth penalty. Here, the authors show that overexpression of Pag-miR408 in poplar leads to enhanced saccharification efficiency and growth in both laboratory and field conditions, and laccase genes are the targets of Pag-miR408 .

The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome-editing approaches to develop improved crops 1 , 2 . Often considered to be genome ‘parasites’, transposable elements (TEs) evolved to insert their DNA seamlessly into genomes 3 – 5 . Eukaryotic TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type 6 – 9 . Here we developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the natural ability of the TE to precisely excise and insert into the genome. Inspired by CRISPR-associated transposases that target transposition in a programmable manner in bacteria 10 – 12 , we fused the rice Pong transposase protein to the Cas9 or Cas12a programmable nucleases. We demonstrated sequence-specific targeted insertion (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and a gene expression cassette into the genome of the model plant Arabidopsis . We then translated this system into soybean—a major global crop in need of targeted insertion technology. We have engineered a TE ‘parasite’ into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes. Fusion of rice Pong transposase to the Cas9 or Cas12a programmable nucleases provides sequence-specific targeted insertion of enhancer elements, an open reading frame and gene expression cassette into the genome of the model plant Arabidopsis and crop soybean .

Stripe (yellow) rust, caused by Puccinia striiformis f. sp. tritici ( Pst ), can significantly affect wheat production. Cloning resistance genes is critical for efficient and effective breeding of stripe rust resistant wheat cultivars. One resistance gene ( Yr10 CG ) underlying the Pst resistance locus Yr10 has been cloned. However, following haplotype and linkage analyses indicate the presence of additional Pst resistance gene(s) underlying/near Yr10 locus. Here, we report the cloning of the Pst resistance gene YrNAM in this region using the method of sequencing trait-associated mutations (STAM). YrNAM encodes a non-canonical resistance protein with a NAM domain and a ZnF-BED domain. We show that both domains are required for resistance. Transgenic wheat harboring YrNAM gene driven by its endogenous promoter confers resistance to stripe rust races CYR32 and CYR33. YrNAM is an ancient gene and present in wild wheat species Aegilops longissima and Ae. sharonensis ; however, it is absent in most wheat cultivars, which indicates its breeding value. Wheat stripe (yellow) rust is a devastating disease posing a threat to global production. Here, the authors report the cloning of a wheat stripe rust resistance gene encoding a protein with a NAM domain and a ZnF-BED domain using a strategy called sequencing trait-associated mutations (STAM).

There is an expanding need to modify plant genomes to create new plant germplasm that advances both basic and applied plant research. Most current methods for plant genome modification involve regenerating plants from genetically modified cells in tissue culture, which is technically challenging, expensive and time consuming, and works with limited plant species or genotypes. Herein, we describe two Agrobacterium -based methods for creating genetic modifications on either sterilely grown or soil-grown Nicotiana benthamiana plants. These methods use developmental regulators (DRs), gene products that influence cell division and differentiation, to induce de novo meristems. Genome editing reagents, such as the RNA-guided endonuclease Cas9, may be co-delivered with the DRs to create shoots that transmit edits to the next generation. One method, called fast-treated Agrobacterium co-culture (Fast-TrACC), delivers DRs to seedlings grown aseptically; meristems that produce shoots and ultimately whole plants are induced. The other approach, called direct delivery (DD), involves delivering DRs to soil-grown plants from which existing meristems have been removed; the DRs promote the formation of new shoots at the wound site. With either approach, if transgene cassettes and/or gene editing reagents are provided, these induced, de novo meristems may be transgenic, edited or both. These two methods offer alternative approaches for generating novel plant germplasm that are cheaper and less technically challenging and take less time than standard approaches. The whole procedure from transfer DNA (T-DNA) assembly to recovery of edited plants can be completed in ~70 d for both DD and Fast-TrACC. This protocol comprises two Agrobacterium -based methods (direct delivery and fast-treated Agrobacterium co-culture) that use developmental regulators to induce de novo meristems to create genetic modifications on either sterilely grown or soil-grown Nicotiana benthamiana plants.

The plant enzyme Rubisco is the main enzyme converting atmospheric carbon dioxide into biological compounds, however, this enzymatic process is inefficient in vascular plants; this study demonstrates that tobacco plants can be engineered to fix carbon with a faster cyanobacterial Rubisco, thus potentially improving plant photosynthesis. Introducing algal Rubisco into a crop plant Rubisco — a major enzyme assimilating atmospheric CO 2 into the biosphere — is an important target for efforts to improve the photosynthetic efficiency of plants. These authors successfully engineered tobacco plants containing a functioning Rubisco from a cyanobacterium. The cyanobacterial (photosynthetic blue–green algae) enzyme has a greater catalytic rate than any 'C3' plant. The lines generated here pave the way for future addition of the remaining components of the cyanobacterial CO 2 -concentrating mechanism, an important step towards enhancing photosynthetic efficiency and improving crop yields. In photosynthetic organisms, d -ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating atmospheric CO 2 into the biosphere 1 . Owing to the wasteful oxygenase activity and slow turnover of Rubisco, the enzyme is among the most important targets for improving the photosynthetic efficiency of vascular plants 2 , 3 . It has been anticipated that introducing the CO 2 -concentrating mechanism (CCM) from cyanobacteria into plants could enhance crop yield 4 , 5 , 6 . However, the complex nature of Rubisco’s assembly has made manipulation of the enzyme extremely challenging, and attempts to replace it in plants with the enzymes from cyanobacteria and red algae have not been successful 7 , 8 . Here we report two transplastomic tobacco lines with functional Rubisco from the cyanobacterium Synechococcus elongatus PCC7942 (Se7942). We knocked out the native tobacco gene encoding the large subunit of Rubisco by inserting the large and small subunit genes of the Se7942 enzyme, in combination with either the corresponding Se7942 assembly chaperone, RbcX, or an internal carboxysomal protein, CcmM35, which incorporates three small subunit-like domains 9 , 10 . Se7942 Rubisco and CcmM35 formed macromolecular complexes within the chloroplast stroma, mirroring an early step in the biogenesis of cyanobacterial β-carboxysomes 11 , 12 . Both transformed lines were photosynthetically competent, supporting autotrophic growth, and their respective forms of Rubisco had higher rates of CO 2 fixation per unit of enzyme than the tobacco control. These transplastomic tobacco lines represent an important step towards improved photosynthesis in plants and will be valuable hosts for future addition of the remaining components of the cyanobacterial CCM, such as inorganic carbon transporters and the β-carboxysome shell proteins 4 , 5 , 6 .

Bacterial cytidine deaminase fused to the DNA binding domains of transcription activator-like effector nucleases was recently reported to transiently substitute a targeted C to a T in mitochondrial DNA of mammalian cultured cells 1 . We applied this system to targeted base editing in the Arabidopsis thaliana plastid genome. The targeted Cs were homoplasmically substituted to Ts in some plantlets of the T 1 generation and the mutations were inherited by their offspring independently of their nuclear-introduced vectors. This study used bacterial cytidine deaminase fused to the DNA binding domains of transcription activator-like effector nucleases to enable targeted base editing in the Arabidopsis thaliana plastid genome, generating T 1 plants with inheritable homoplasmic mutations.