Alfalfa (Medicago sativa. L.) is a globally significant autotetraploid legume forage crop. However, despite its importance, establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge. In this study, we pioneered the development of a highly effective ultrasonic-assisted leaf disc transformation system for Gongnong 1 alfalfa, a variety widely cultivated in Northeast China. Subsequently, we created a single transcript CRISPR/Cas9 (CRISPR_2.0) toolkit, incorporating multiplex gRNAs, designed for gene editing in Gongnong 1. Both Cas9 and gRNA scaffolds were under the control of the Arabidopsis ubiquitin-10 promoter, a widely employed polymerase II constitutive promoter known for strong transgene expression in dicots. To assess the toolkit’s efficiency, we targeted PALM1, a gene associated with a recognizable multifoliate phenotype. Utilizing the CRISPR_2.0 toolkit, we directed PALM1 editing at two sites in the wild-type Gongnong 1. Results indicated a 35.1% occurrence of editing events all in target 2 alleles, while no mutations were detected at target 1 in the transgenic-positive lines. To explore more efficient sgRNAs, we developed a rapid, reliable screening system based on Agrobacterium rhizogenes-mediated hairy root transformation, incorporating the visible reporter MtLAP1. This screening system demonstrated that most purple visible hairy roots underwent gene editing. Notably, sgRNA3, with an 83.0% editing efficiency, was selected using the visible hairy root system. As anticipated, tetra-allelic homozygous palm1 mutations exhibited a clear multifoliate phenotype. These palm1 lines demonstrated an average crude protein yield increase of 21.5% compared to trifoliolate alfalfa. Our findings highlight the modified CRISPR_2.0 system as a highly efficient and robust gene editing tool for autotetraploid alfalfa.

Amylose content, the key determinant of rice eating and cooking quality, is regulated primarily by the Waxy (Wx) gene. We adjusted the amylose content and transparency of semi-glutinous japonica rice carrying the Wx^mp allele by genome editing of upstream open reading frame 6 (uORF6) of Wx.

The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement. Currently available genome-editing tools have a limited number of targets, restricting their application in genetic research. In this study, we developed a novel CRISPR/Cas9 plant ultra-multiplex genome editing system consisting of two template vectors, eight donor vectors, four destination vectors, and one primer-design software package. By combining the advantages of Golden Gate cloning to assemble multiple repetitive fragments and Gateway recombination to assemble large fragments and by changing the structure of the amplicons used to assemble sgRNA expression cassettes, the plant ultra-multiplex genome editing system can assemble a single binary vector targeting more than 40 genomic loci. A rice knockout vector containing 49 sgRNA expression cassettes was assembled and a high co-editing efficiency was observed. This plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.

Soybean is the primary source of plant protein for humans. Owing to the indigestibility of the raffinose family of oligosaccharides (RFO), raffinose and stachyose are considered anti-nutritive factors in soybean seeds. Low-RFO soybean cultivars are generated by mutagenesis of RFO biosynthesis genes, but the carbohydrate profiles invite further modification to lower RFOs. This study employed a pooled multiplex genome editing approach to target four seed-specifically expressed genes mediating RFO biosynthesis, encoding three raffinose synthases (RS2, RS3, and RS4) and one stachyose synthase. In T₁ progeny, rs2/rs3 and rs4/sts homozygous double mutants and a rs2/rs3/rs4/sts quadruple mutant (rfo-4m) were characterized. The rs2/rs3 mutant showed reduced raffinose and stachyose contents, but the rs4/sts mutant showed only reduced stachyose in seeds. The RFO contents in the rfo-4m mutant were almost eliminated. Metabolomic analysis showed that the mutation of four RFO biosynthesis genes led to a shift of metabolic profile in the seeds, including the accumulation of several oligosaccharides-related metabolites. These mutants could contribute to precision breeding of soybean cultivars for soy food production.

A fast and efficient recognition method of transgenic lines will greatly improve detection efficiency and reduce cost. In this study, we successfully identified the transgenic soybean plants by the color. We isolated a GmW1 gene encoding a flavonoid 3′5′-hydroxylase from a soybean cultivar ZH42 (purple flower). We found that purple flowers occurred in the overexpression lines in the Jack and Williams 82 backgrounds (white flower). All plants with purple flowers were positive, and this trait seems stably inherited in the offspring. We have also obtained the editing plants, which were classified into three types according to the different flower colors appeared. We analyzed the phenotype and the homozygous types of the T₁ mutants. We also found that a correspondence between flower color and stem color. This study provides a visible color reporter on soybean transformation. It can be quickly and early to identify the transgenic soybean plants by stem color of seedlings, which substantially reduces the amount of labor and cost.

Increasing population and consumption in our planet is placing unprecedented challenges on agriculture for meeting food security and sustainability needs. Meanwhile, the adaptation of modern agricultural techniques is central to minimize extensive losses due to abiotic stresses under global climate change. Among these agricultural technology systems, crop breeding is the core node of all technologies and is finally reflected in crops and their products. Crop breeding deals with the creation and selection of the desired variation in target varieties with improved yield, quality and tolerance to abiotic and biotic stresses. Random mutagenesis using physical, chemical, and biological approaches has been limited by the availability of desirable mutant alleles.

Grain size is one of the most important factors affecting rice grain quality and yield, and attracts great attention from molecular biologists and breeders. In this study, we engineered a CRISPR/Cas9 system targeting the miR396 recognition site of the rice GS2 gene, which encodes growth-regulating factor 4 (OsGRF4) and regulates multiple agronomic traits including grain size, grain quality, nitrogen use efficiency, abiotic stress response, and seed shattering. In contrast to most previous genome editing efforts in which indel mutations were chosen to obtain null mutants, a mutant named GS2^E carrying an in-frame 6-bp deletion and 1-bp substitution within the miR396-targeted sequence was identified. GS2^E plants showed increased expression of GS2 in consistent with impaired repression by miR396. As expected, the gain-of-function GS2^E mutant exhibited multiple beneficial traits including increased grain size and yield and bigger grain length/width ratio. Thousand grain weight and grain yield per plant of GS2^E plants were increased by 23.5% and 10.4%, respectively. These improved traits were passed to hybrids in a semi-dominant way, suggesting that the new GS2^E allele has great potential in rice improvement. Taken together, we report new GS2 germplasm and describe a novel gene-editing strategy that can be widely employed to improve grain size and yield in rice. This trait-improvement strategy could be applied to other genes containing miRNA target sites, in particular the conserved miR396-GRF/GIF module that governs plant growth, development and environmental response.

Sequence-specific nucleases (SSN) that generate double-stranded DNA breaks (DSBs) in genes of interest are the key to site-specific genome editing in plants. Genome editing has developed into one method of reducing undesirable traits in crops by the induction of knockout mutations. Different SSN-mediated genome-editing systems, including LAGLIDADG homing endonucleases or meganucleases, zinc-finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats, are emerging as robust tools for introducing functional mutations in polyploid crops including citrus, wheat, cotton, soybean, rapeseed, potato, grapes, Camelina sativa, dandelion, and tobacco. The approach utilizes knowledge of biological mechanisms for targeted induction of DSBs and their error-prone repair, allowing highly specific changes at designated genome loci. In this review, we briefly describe genome-editing technologies and their application to genetic improvement of polyploid crops.

Single-guide RNA (sgRNA) is one of the two core components of the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome-editing technology. We established an in vitro Traffic Light Reporter (TLR) system, which is designated as the same colors as traffic lights such as green, red and yellow were produced in cells. The TLR can be readily used in maize mesophyll protoplast for a quick test of promoter activity. The TLR assay indicates the variation in transcription activities of the seven Pol III promoters, from 3.4% (U6-1) to over 21.0% (U6-6). The U6-2 promoter, which was constructed to drive sgRNA expression targeting the ZmWx1 gene, yielded mutation efficiencies ranging from 48.5% to 97.1%. Based on the reported and unpublished data, the in vitro TLR assay results were confirmed to be a readily system and may be extended to other plant species amenable to efficient genome editing via CRISPR/Cas. Our efforts provide an efficient method of identifying native Pol III-recognized promoters for RNA guide-based genome-editing systems in maize.

Genome editing is one of the most promising biotechnologies to improve crop performance. Common wheat is a staple food for mankind. In the past few decades both basic and applied research on common wheat has lagged behind other crop species due to its complex, polyploid genome and difficulties in genetic transformation. Recent breakthroughs in wheat transformation permit a revolution in wheat biotechnology. In this review, we summarize recent progress in wheat genetic transformation and its potential for wheat improvement. We then review recent progress in plant genome editing, which is now readily available in wheat. We also discuss measures to further increase transformation efficiency and potential applications of genome editing in wheat. We propose that, together with a high quality reference genome, the time for efficient genetic engineering and functionality studies in common wheat has arrived.

The CRISPR/Cas9 technology is evolved from a type II bacterial immune system and represents a new generation of targeted genome editing technology that can be applied to nearly all organisms. Site-specific modification is achieved by a single guide RNA (usually about 20 nucleotides) that is complementary to a target gene or locus and is anchored by a protospacer-adjacent motif. Cas9 nuclease then cleaves the targeted DNA to generate double-strand breaks (DSBs), which are subsequently repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms. NHEJ may introduce indels that cause frame shift mutations and hence the disruption of gene functions. When combined with double or multiplex guide RNA design, NHEJ may also introduce targeted chromosome deletions, whereas HDR can be engineered for target gene correction, gene replacement, and gene knock-in. In this review, we briefly survey the history of the CRISPR/Cas9 system invention and its genome-editing mechanism. We also describe the most recent innovation of the CRISPR/Cas9 technology, particularly the broad applications of modified Cas9 variants, and discuss the potential of this system for targeted genome editing and modification for crop improvement.