Isolation and Functional Characterization of a Constitutive Promoter in Upland Cotton (Gossypium hirsutum L.).

Multiple cis-acting elements are present in promoter sequences that play critical regulatory roles in gene transcription and expression. In this study, we isolated the cotton FDH (Fiddlehead) gene promoter (pGhFDH) using a real-time reverse transcription-PCR (qRT-PCR) expression analysis and performed a cis-acting elements prediction analysis. The plant expression vector pGhFDH::GUS was constructed using the Gateway approach and was used for the genetic transformation of Arabidopsis and upland cotton plants to obtain transgenic lines. Histochemical staining and a ß-glucuronidase (GUS) activity assay showed that the GUS protein was detected in the roots, stems, leaves, inflorescences, and pods of transgenic Arabidopsis thaliana lines. Notably, high GUS activity was observed in different tissues. In the transgenic lines, high GUS activity was detected in different tissues such as leaves, stalks, buds, petals, androecium, endosperm, and fibers, where the pGhFDH-driven GUS expression levels were 3-10-fold higher compared to those under the CaMV 35S promoter at 10-30 days post-anthesis (DPA) during fiber development. The results indicate that pGhFDH can be used as an endogenous constitutive promoter to drive the expression of target genes in various cotton tissues to facilitate functional genomic studies and accelerate cotton molecular breeding.

Precise fine-turning of GhTFL1 by base editing tools defines ideal cotton plant architecture.

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 GhTFL1L86P and GhTFL1K53G+S78G mutations, we find that the GhTFL1L86P 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.

Robust CRISPR/Mb2Cas12a genome editing tools in cotton plants.

The efficiency and accuracy of the CRISPR/Mb2Cas12a system were demonstrated in cotton, achieving an efficiency of over 90% at target sites. Notably, Mb2Cas12a exhibited significant tolerance under different temperatures ranging from 22°C to 32°C. Additionally, the Mb2Cas12a system revealed effective editing at more relaxed VTTV PAM sites in the cotton genome, which expanded the genome editing range by approximately 2.6-fold than the wide-type LbCas12a. Finally, a multiplex genome editing system was also developed based on Mb2Cas12a, enabling simultaneous editing of eight target sites using a single crRNA cassette.

Construction of Host Plant Insect-Resistance Mutant Library by High-Throughput CRISPR/Cas9 System and Identification of A Broad-Spectrum Insect Resistance Gene.

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.

Highly Efficient Genome Editing Using Geminivirus-Based CRISPR/Cas9 System in Cotton Plant.

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.

Herbicide Resistance: Another Hot Agronomic Trait for Plant Genome Editing.

Weeds have continually interrupted crop plants since their domestication, leading to a greater yield loss compared to diseases and pests that necessitated the practice of weed control measures. The control of weeds is crucial to ensuring the availability of sufficient food for a rapidly increasing human population. Chemical weed control (herbicides) along with integrated weed management (IWM) practices can be the most effective and reliable method of weed management programs. The application of herbicides for weed control practices calls for the urgency to develop herbicide-resistant (HR) crops. Recently, genome editing tools, especially CRISPR-Cas9, have brought innovation in genome editing technology that opens up new possibilities to provide sustainable farming in modern agricultural industry. To date, several non-genetically modified (GM) HR crops have been developed through genome editing that can present a leading role to combat weed problems along with increasing crop productivity to meet increasing food demand around the world. Here, we present the chemical method of weed control, approaches for herbicide resistance development, and possible advantages and limitations of genome editing in herbicide resistance. We also discuss how genome editing would be effective in combating intensive weed problems and what would be the impact of genome-edited HR crops in agriculture.