Explore the vast range of titles available.

Background CRISPR/Cas-derived base editor enables precise editing of target sites and has been widely used for basic research and crop genetic improvement. However, the editing efficiency of base editors at different targets varies greatly. Results Here, we develop a set of highly efficient base editors in cotton plants. GhABE8e, which is fused to conventional nCas9, exhibits 99.9% editing efficiency, compared to GhABE7.10 with 64.9%, and no off-target editing is detected. We further replace nCas9 with dCpf1, which recognizes TTTV PAM sequences, to broaden the range of the target site. To explore the functional divergence of TERMINAL FLOWER 1 (TFL1), we edit the non-coding and coding regions of GhTFL1 with 26 targets to generate a comprehensive allelic population including 300 independent lines in cotton. This allows hidden pleiotropic roles for GhTFL1 to be revealed and allows us to rapidly achieve directed domestication of cotton and create ideotype germplasm with moderate height, shortened fruiting branches, compact plant, and early-flowering. Further, by exploring the molecular mechanism of the GhTFL1 L86P and GhTFL1 K53G+S78G mutations, we find that the GhTFL1 L86P mutation weakens the binding strength of the GhTFL1 to other proteins but does not lead to a complete loss of GhTFL1 function. Conclusions This strategy provides an important technical platform and genetic information for the study and creation of ideal plant architecture.

Background Base editors (BEs) display diverse applications in a variety of plant species such as Arabidopsis, rice, wheat, maize, soybean, and cotton, where they have been used to mediate precise base pair conversions without the collateral generation of undesirable double-stranded breaks (DSB). Studies of single-nucleotide polymorphisms (SNPs) underpinning plant traits are still challenging, particularly in polyploidy species where such SNPs are present in multiple copies, and simultaneous modification of all alleles would be required for functional analysis. Allotetraploid cotton has a number of homoeologous gene pairs located in the A and D sub-genomes with considerable SNPs, and it is desirable to develop adenine base editors (ABEs) for efficient and precise A-to-G single-base editing without DSB in such complex genome. Results We established various ABE vectors based on different engineered adenosine deaminase (TadA) proteins fused to Cas9 variants (dCas9, nCas9), enabling efficient A to G editing up to 64% efficiency on-target sites of the allotetraploid cotton genome. Comprehensive analysis showed that GhABE7.10n exhibited the highest editing efficiency, with the main editing sites specifically located at the position A5 (counting the PAM as positions 21–23). Furthermore, DNA and RNA off-target analysis of cotton plants edited with GhABE7.10n and GhABE7.10d by whole genome and whole-transcriptome sequencing revealed no DNA off-target mutations, while very low-level RNA off-target mutations were detected. A new base editor, namely GhABE7.10dCpf1 (7.10TadA + dCpf1), that recognizes a T-rich PAM, was developed for the first time. Targeted A-to-G substitutions generated a single amino acid change in the cotton phosphatidyl ethanolamine-binding protein ( GhPEBP ), leading to a compact cotton plant architecture, an ideotype for mechanized harvesting of modern cotton production. Conclusions Our data illustrate the robustness of adenine base editing in plant species with complex genomes, which provides efficient and precise toolkit for cotton functional genomics and precise molecular breeding.

[...]different types of genome editing tools are desirable to improve the functional genomic research in cotton. A previous work has shown that tRNA‐sgRNA transcription unit is very effective to enhance the sgRNA transcription in CRISPR/Cas9 system for cotton genome editing (Wang et al., ). [...]we designed the crRNA following our previous work and conducted a vector named pGhRBE3‐Cpf1‐GhCLA1 targeting GhCLA1 gene (Figure a). The editing window and profiling of CRISPR/Cpf1 system in the edited T0 plants were systematically analysed in this report. Since no target mutation occurs preceding the PAM sequence and within the PAM sequence, we investigated the editing downstream of the PAM sequence. Deletion size and proportion of each target mutation in T0 plants were also analysed and are shown in Figure e. The deletion sizes ranged from 3 to 28 bp in length, and the deletion size of majority is from 5 to 12 bp (Figure e), which are larger than the average deletion size (1–5 bp) induced by CRISPR/Cas9 in cotton genome (Wang et al., ). [...]we speculate that the CRISPR/Cpf1 system prefers to generate large size deletions in cotton genome editing.

Insects pose significant challenges in cotton‐producing regions. Here, they describe a high‐throughput CRISPR/Cas9‐mediated large‐scale mutagenesis library targeting endogenous insect‐resistance‐related genes in cotton. This library targeted 502 previously identified genes using 968 sgRNAs, generated ≈2000 T0 plants and achieved 97.29% genome editing with efficient heredity, reaching upto 84.78%. Several potential resistance‐related mutants (10% of 200 lines) their identified that may contribute to cotton‐insect molecular interaction. Among these, they selected 139 and 144 lines showing decreased resistance to pest infestation and targeting major latex‐like protein 423 (GhMLP423) for in‐depth study. Overexpression of GhMLP423 enhanced insect resistance by activating the plant systemic acquired resistance (SAR) of salicylic acid (SA) and pathogenesis‐related (PR) genes. This activation is induced by an elevation of cytosolic calcium [Ca2+]cyt flux eliciting reactive oxygen species (ROS), which their demoted in GhMLP423 knockout (CR) plants. Protein‐protein interaction assays revealed that GhMLP423 interacted with a human epidermal growth factor receptor substrate15 (EPS15) protein at the cell membrane. Together, they regulated the systemically propagating waves of Ca2+ and ROS, which in turn induced SAR. Collectively, this large‐scale mutagenesis library provides an efficient strategy for functional genomics research of polyploid plant species and serves as a solid platform for genetic engineering of insect resistance. A high‐throughput screening strategy for cotton endogenous insect‐resistance‐related genes is established by a CRISPR/Cas9‐mediated large‐scale mutant library. It identifies promising genes related to plant innate insect‐resistance, including major latex‐like protein 423 (GhMLP423). GhMLP423 enhanced plant immunity to chewing and sap‐sacking insects via the induction of GhEPS15 that initiates the systemic acquired resistance elicited by the systemically propagating waves of Ca2+‐mediated ROS signaling.

N6‐methyladenosine (m6A) is the most prevalent internal modification of mRNA and plays an important role in regulating plant growth. However, there is still a lack of effective tools to precisely modify m6A sites of individual transcripts in plants. Here, programmable m6A editing tools are developed by combining CRISPR/dCas13(Rx) with the methyltransferase GhMTA (Targeted RNA Methylation Editor, TME) or the demethyltransferase GhALKBH10 (Targeted RNA Demethylation Editor, TDE). These editors enable efficient deposition or removal of m6A modifications at targeted sites of endo‐transcripts GhECA1 and GhDi19 within a broad editing window ranging from 0 to 46 nt. TDE editor significantly decreases m6A levels by 24%–76%, while the TME editor increases m6A enrichment, ranging from 1.37‐ to 2.51‐fold. Furthermore, installation and removal of m6A modifications play opposing roles in regulating GhECA1 and GhDi19 mRNA transcripts, which may be attributed to the fact that their m6A sites are located in different regions of the genes. Most importantly, targeting the GhDi19 transcript with TME editor plants results in a significant increase in root length and enhanced drought resistance. Collectively, these m6A editors can be applied to study the function of specific m6A modifications and have the potential for future applications in crop improvement. TDE and TME editors can efficiently deposit and remove the m6A modification at targeted sites, thereby altering mRNA abundance and potentially influencing downstream phenotypes in individual transcripts. These m6A editors operate across a broad editing window and exhibit high specificity for off‐target effects, which is essential for investigating the roles of specific m6A modifications in plants.

Upland cotton (Gossypium hirsutum), an allotetraploid, contains At- and Dt- subgenome and most genes have multiple homologous copies, which pose a huge challenge to investigate genes’ function due to the functional redundancy. Therefore, it is of great significance to establish effective techniques for the functional genomics in cotton. In this study, we tested two novel genome editing vectors and compared them with the CRISPR/Cas9 system (pRGEB32-GhU6.7) developed in our laboratory previously. In the first new vector, the sgRNA transcription unite was constructed into the replicon (LIR-Donor-SIR-Rep-LIR) of the bean yellow dwarf virus (BeYDV) and named as pBeYDV-Cas9-KO and in the second vector, the ubiquitin promoter that drives Cas9 protein was replaced with a constitutive CaMV 35S promoter and defined as pRGEB32-35S. The results from transgenic cotton calli/plants revealed that pBeYDV-Cas9-KO vector showed the highest editing efficiency of GhCLA1 in At and Dt subgenomes edited simultaneously up to 73.3% compared to the 44.6% of pRGEB32-GhU6.7 and 51.2% of pRGEB32-35S. The editing efficiency of GhCLA1 in At and Dt subgenome by pBeYDV-Cas9-KO was 85.7% and 97.2%, respectively, whereas the efficiency by pRGEB32-GhU6.7 and pRGEB32-35S vectors was 67.7%, 86.5%, 84%, and 87.2%, respectively. The editing profile of pBeYDV-Cas9-KO was mainly composed of fragment deletion, accounting for 84.0% and ranging 1–10 bp in length. The main editing sites are located at positions 11–17 upstream of PAM site. The off-target effects were not detected in all potential off-target sites. Taken together, the pBeYDV-Cas9-KO system has high editing efficiency and specificity with wide editing range than the traditional CRISPR/Cas9 system, which provides a powerful tool for cotton functional genomics research and molecular breeding.

Hybrid breeding has provided an impetus to the process and achievement of a higher yield and quality of crops. Interspecific hybridization is critical for resolving parental genetic diversity bottleneck problems. The reciprocal interspecific hybrids and their parents ( Gossypium hirsutum and Gossypium barbadense ) have been applied in this study to elucidate the transcription regulatory mechanism of early biomass heterosis. Phenotypically, the seed biomass, plant height over parent heterosis, leaf area over parent heterosis, and fresh and dry biomass were found to be significantly higher in hybrids than in parents. Analysis of leaf areas revealed that the one-leaf stage exhibits the most significant performance in initial vegetative growth vigor and larger leaves in hybrids, increasing the synthesis of photosynthesis compounds and enhancing photosynthesis compound synthesis. Comparative transcriptome analysis showed that transgressive down-regulation (TDR) is the main gene expression pattern in the hybrids ( G. hirsutum × G. barbadense , HB), and it was found that the genes of photosystem I and Adenosine triphosphate (ATP)-binding may promote early growth vigor. Transgressive up-regulation (TUR) is the major primary gene expression pattern in the hybrids ( G. barbadense × G. hirsutum , BH), and photosystem II-related genes mediated the performance of early biomass heterosis. The above results demonstrated that overdominance mediates biomass heterosis in interspecific hybrid cotton and the supervisory mechanism divergence of hybrids with different females. Photosynthesis and other metabolic process are jointly involved in controlling early biomass heterosis in interspecific hybrid cotton. The expression pattern data of transcriptome sequencing were supported using the qRT-PCR analysis. Our findings could be useful in theoretical and practical studies of early interspecific biomass heterosis, and the results provide potential resources for the theoretical and applied research on early interspecific biomass heterosis.

Summary The CRISPR/Cas9 and Cas12a (Cpf1) tools have been used on a large scale for genome editing. A new effector with a single nuclease domain, a relatively small size, low‐frequency off‐target effects and cleavage capability under high temperature has been recently established and designated CRISPR/Cas12b (C2c1). Cas12b has also shown temperature inducibility in mammalian systems. Therefore, this system is potentially valuable for editing the genomes of plant species, such as cotton, that are resistant to high temperatures. Using this new system, mutants of upland cotton were successfully generated following Agrobacterium‐mediated genetic transformation under a range of temperatures. Transformants (explants infected by Agrobacterium) exposed to 45 °C for 4 days showed the highest editing efficiency. No off‐target mutation was detected by whole‐genome sequencing. Genome edits by AacCas12b in T0 generation were faithfully passed to the T1 generation. Taken together, CRISPR/Cas12b is therefore an efficient and precise tool for genome editing in cotton plants.

Cotton (Gossypium hirsutum) is an allotetraploid species and a typical thermophilic crop that can survive and grow well under temperatures up to 45 °C. CRISPR/LbCpf1 (LbCas12a) is a temperature‐sensitive system for plant genome editing (Malzahn et al., 2019) and has been successfully applied in species such as rice, soya bean, tobacco, maize and cotton (Lee et al., 2019; Li et al., 2018; Tang et al., 2017; Xu et al., 2019). In order to improve LbCpf1 efficiency and determine the optimum temperature for cotton genome editing, we investigated the effects of different temperatures on LbCpf1 activity and genome editing efficiency. [...]we created nontransgenic and glandless cotton plants with seeds free of gossypol, representing a valuable germplasm resource for cotton breeding. With the increase of temperature, the bleaching phenotype of leaves becomes more obvious in T1 LbCpf1‐GhCLA1 transgenic plants grown in the field. (b) Phenotypes of T1 cotton plants with the target mutations in the GhCLA1 gene, grown at different temperatures.

Summary Zanthoxylum armatum and Zanthoxylum bungeanum, known as ‘Chinese pepper’, are distinguished by their extraordinary complex genomes, phenotypic innovation of adaptive evolution and species‐special metabolites. Here, we report reference‐grade genomes of Z. armatum and Z. bungeanum. Using high coverage sequence data and comprehensive assembly strategies, we derived 66 pseudochromosomes comprising 33 homologous phased groups of two subgenomes, including autotetraploid Z. armatum. The genomic rearrangements and two whole‐genome duplications created large (~4.5 Gb) complex genomes with a high ratio of repetitive sequences (>82%) and high chromosome number (2n = 4x = 132). Further analysis of the high‐quality genomes shed lights on the genomic basis of involutional reproduction, allomones biosynthesis and adaptive evolution in Chinese pepper, revealing a high consistent relationship between genomic evolution, environmental factors and phenotypic innovation. Our study provides genomic resources and new insights for investigating diversification and phenotypic innovation in Chinese pepper, with broader implications for the protection of plants under severe environmental changes.