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(A) Golden Gate cloning principle applied for assembly of dTALEs. Plasmids encoding selected repeat modules (an example with only two modules, R1 and R2, is shown here due to space limitation) are mixed in one tube together with BsaI, T4 DNA ligase and the destination vector (containing a lacZα fragment for blue-white selection). Assembly of R1 and R2 using BsaI and ligase gives rise to a plasmid lacking the initial BsaI sites, but containing a block of assembled repeats flanked by two BpiI sites. The two BpiI sites allow release of the assembled repeats as one block for the second step of cloning. fs, fusion site. (B) Structure of AvrBs3. AvrBs3 contains a central region with 17 direct repeats (light grey boxes) flanked by a thymidine-specific repeat (repeat 0) and a half repeat (repeat 17.5, both flanking repeats shown as dark grey boxes). Two nuclear localization sequences (NLS, black bars) and a transcription activation domain (AD) are located in the C-terminal region. One representative 34 aa repeat is shown, with the RVD of the NI type highlighted in grey. (C) RVD types and their specificities. (D) Set of 68 repeat modules, with 4 modules with different specificities for each of the 17 repeat positions. Repeat modules are flanked by two BsaI sites with fusion sites selected from the codon-optimized sequence of AvrBs3 (see Supporting Information S1). Sets of five (for repeats 13–17) or six (for repeats 1–6 and 7–12) selected repeat modules are preassembled via BsaI into preassembly vectors (pL1-TA1 to 3). Preassembled repeat blocks are then combined in the final destination vector (pL2-TA) using a second BpiI-based Golden Gate cloning reaction. Construction of dTALE-1 is shown as an example.
Source publication
Generation of customized DNA binding domains targeting unique sequences in complex genomes is crucial for many biotechnological applications. The recently described DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas consists of a series of repeats arranged in tandem, each repeat binding a nucleotide of the tar...
Citations
... The entire gene and region were compared in Geneious, and 600-800-bp upstream of the 13 genes was selected, including any annotated 5 0 UTR. These promoter sequences then were synthesized with 5 0 and 3 0 4-bp MoClo promoter overhangs flanked by BsaI recognition sites for Golden Gate cloning as promoter+5 0 UTR standard parts (Weber et al., 2011). Any BpiI and BsaI enzyme recognition sites within the promoter sequence were removed before synthesis. ...
Plant resistance (R) and pathogen avirulence (Avr) gene interactions play a vital role in pathogen resistance. Efficient molecular screening tools for crops lack far behind their model organism counterparts, yet they are essential to rapidly identify agriculturally important molecular interactions that trigger host resistance.
Here, we have developed a novel wheat protoplast assay that enables efficient screening of Avr/R interactions at scale. Our assay allows access to the extensive gene pool of phenotypically described R genes because it does not require the overexpression of cloned R genes. It is suitable for multiplexed Avr screening, with interactions tested in pools of up to 50 Avr candidates.
We identified Avr/R‐induced defense genes to create a promoter‐luciferase reporter. Then, we combined this with a dual‐color ratiometric reporter system that normalizes read‐outs accounting for experimental variability and Avr/R‐induced cell death. Moreover, we introduced a self‐replicative plasmid reducing the amount of plasmid used in the assay.
Our assay increases the throughput of Avr candidate screening, accelerating the study of cellular defense signaling and resistance gene identification in wheat. We anticipate that our assay will significantly accelerate Avr identification for many wheat pathogens, leading to improved genome‐guided pathogen surveillance and breeding of disease‐resistant crops.
... The introduction of variants at this enhancer disrupted promoter interaction with TNFAIP3 and ultimately led to the downregulation of the downstream autoimmune gene NF-κB. While TALENs allow for targeted genetic perturbations, they are too difficult to scale for genetic screens (Morbitzer et al., 2011;Reyon et al., 2012;Weber et al., 2011). In contrast, CRISPR/Cas9 has been widely adopted to endogenously perturb genes (Jinek et al., 2012). ...
Despite ground-breaking genetic studies that have identified thousands of risk variants for developmental diseases, how these variants lead to molecular and cellular phenotypes remains a gap in knowledge. Many of these variants are non-coding and occur at enhancers, which orchestrate key regulatory programs during development. The prevailing paradigm is that non-coding variants alter the activity of enhancers, impacting gene expression programs, and ultimately contributing to disease risk. A key obstacle to progress is the systematic functional characterization of non-coding variants at scale, especially since enhancer activity is highly specific to cell type and developmental stage. Here, we review the foundational studies of enhancers in developmental disease and current genomic approaches to functionally characterize developmental enhancers and their variants at scale. In the coming decade, we anticipate systematic enhancer perturbation studies to link non-coding variants to molecular mechanisms, changes in cell state, and disease phenotypes.
... CRISPR sgRNAs were designed on CRISPOR [75]. CRISPR constructs were assembled using the MoClo toolkit and Golden Gate cloning [76,77]. Each sgRNA was expressed under an Arabidopsis U6-26 promoter, and hCas9 was driven by 2 × proCaMV35S as described by Slaman et al. [78]. ...
The moment at which a plant transitions to reproductive development is paramount to its life cycle and is strictly controlled by many genes. The transcription factor SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) plays a central role in this process in Arabidopsis. However, the role of SOC1 in tomato (Solanum lycopersicum) has been sparsely studied. Here, we investigated the function of four tomato SOC1 homologs in the floral transition and inflorescence development. We thoroughly characterized the SOC1-like clade throughout the Solanaceae and selected four tomato homologs that are dynamically expressed upon the floral transition. We show that of these homologs, TOMATO MADS 3 (TM3) and SISTER OF TM3 (STM3) promote the primary and sympodial transition to flowering, while MADS-BOX PROTEIN 23 (MBP23) and MBP18 hardly contribute to flowering initiation in the indeterminate cultivar Moneyberg. Protein–protein interaction assays and whole-transcriptome analysis during reproductive meristem development revealed that TM3 and STM3 interact and share many targets with FRUITFULL (FUL) homologs, including cytokinin regulators. Furthermore, we observed that mutating TM3/STM3 affects inflorescence development, but counteracts the inflorescence-branching phenotype of ful2 mbp20. Collectively, this indicates that TM3/STM3 promote the floral transition together with FUL2/MBP20, while these transcription factors have opposite functions in inflorescence development.
... edu. cn/ CRISP R2) and cloned into the T-DNA vectors that contain the Cas9 gene optimized for plants (Weber et al. 2011). ...
Main conclusionCRISPR/Cas9-mediated Phospholipase C2 knock-out tomato plants are more resistant to Botrytis cinerea than wild-type plants, with less ROS and an increase and reduction of (JA) and (SA)-response marker genes, respectively.AbstractGenome-editing technologies allow non-transgenic site-specific mutagenesis of crops, offering a viable alternative to traditional breeding methods. In this study we used CRISPR/Cas9 to inactivate the tomato Phospholipase C2 gene (SlPLC2). Plant PLC activation is one of the earliest responses triggered by different pathogens regulating plant responses that, depending on the plant–pathogen interaction, result in plant resistance or susceptibility. The tomato (Solanum lycopersicum) PLC gene family has six members, named from SlPLC1 to SlPLC6. We previously showed that SlPLC2 transcript levels increased upon xylanase infiltration (fungal elicitor) and that SlPLC2 participates in plant susceptibility to Botrytis cinerea. An efficient strategy to control diseases caused by pathogens is to disable susceptibility genes that facilitate infection. We obtained tomato SlPLC2-knock-out lines with decreased ROS production upon B. cinerea challenge. Since this fungus requires ROS-induced cell death to proliferate, SlPLC2-knock-out plants showed an enhanced resistance with smaller necrotic areas and reduced pathogen proliferation. Thus, we obtained SlPLC2 loss-of-function tomato lines more resistant to B. cinerea by means of CRISPR/Cas9 genome editing technology.
... Previous lineage analysis predicted that ToBRFV probably evolved on an unknown host other than tomato, and invaded tomato due to a host-shifting event (Maayan et al., 2018). This allowed MP ToBRFV to diverge from MP TMV and MP ToMV in multiple elements, including C68/H67, while (Weber et al., 2011) as described by Hak and Spiegelman (2021). Generation of the vectors TMV-GFP MP-ToBRFV and ToMV MP-ToBRFV , based on the pJL24 (Lindbo, 2007) and ...
The tomato Tm-22 gene was considered to be one of the most durable resistance genes in agriculture, protecting against viruses of the Tobamovirus genus, such as tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV). However, an emerging tobamovirus, tomato brown rugose fruit virus (ToBRFV), has overcome Tm-22 , damaging tomato production worldwide. Tm-22 encodes a nucleotide-binding leucine-rich repeat (NLR) class immune receptor that recognizes its effector, the tobamovirus movement protein (MP). Previously, we found that ToBRFV MP (MPToBRFV ) enabled the virus to overcome Tm-22 -mediated resistance. Yet, it was unknown how Tm-22 remained durable against other tobamoviruses, such as TMV and ToMV, for over 60 years. Here, we show that a conserved cysteine (C68) in the MP of TMV (MPTMV ) plays a dual role in Tm-22 activation and viral movement. Substitution of MPToBRFV amino acid H67 with the corresponding amino acid in MPTMV (C68) activated Tm-22 -mediated resistance. However, replacement of C68 in TMV and ToMV disabled the infectivity of both viruses. Phylogenetic and structural prediction analysis revealed that C68 is conserved among all Solanaceae-infecting tobamoviruses except ToBRFV and localizes to a predicted jelly-roll fold common to various MPs. Cell-to-cell and subcellular movement analysis showed that C68 is required for the movement of TMV by regulating the MP interaction with the endoplasmic reticulum and targeting it to plasmodesmata. The dual role of C68 in viral movement and Tm-22 immune activation could explain how TMV was unable to overcome this resistance for such a long period.
... Over the years, several platforms have been developed to facilitate the assembly of plasmids that encodes TALE repeat arrays. Generally, these methods are grouped as (1) standard restriction enzyme and ligation-based cloning (Huang et al. 2011;Sander et al. 2011); (2) golden gate cloning (Cermak et al. 2011;Weber et al. 2011); and (3) solid-phase assembly (Briggs et al. 2012). The architectures of TALENs such as the length and sequence composition of the N and C terminal TALE-derived sequences are crucial factors while deciding on the type of assembly method. ...
Plants are considered an essential source of numerous therapeutic means for humans. These plant-derived medicines are utilized for various pharmaceutical products including different drug formulations. However, the large-scale production of these plants has become a challenge due to various factors such as susceptibility to diseases, pests, and poor tolerance to abiotic stresses. Even though advances have been made to improve breeding pipelines for medicinal plants, certain bottlenecks have remained. To broaden the horizons of conventional breeding strategies, current genome editing tools are assisting precise gene editing in terms of stress resilience, adaptation, and marketability, in a shorter time frame. This chapter summarizes the utilization of engineered nucleases to transmute the genomic targeted site with an artificially designed and precise controlled genetic circuit in a plant system. In addition, the latest progress in the gene-editing approach including the remarkable instances of the CRISPR system application for stress tolerance in medicinal plants was also highlighted. Lastly, potential threats and future outlooks in using genome-editing tools for medicinal plants are also addressed.
... Likewise, many modular assembly schemes to generate engineered TALEs constructs are in use (Cermak et al., 2011;T. Li et al., 2011;Weber et al., 2011;. ...
Plants produce a large number of secondary metabolites, known as phytometabolites that may be employed as medicines, dyes, poisons, and insecticides in the field of medicine, agriculture, and industrial use, respectively. The rise of genome management approaches has promised a factual revolution in genetic engineering. Targeted genome editing in living entities permits the understanding of the biological systems very clearly, and also sanctions to address a wide‐ranging objective in the direction of improving features of plant and their yields. The last few years have introduced a number of unique genome editing systems, including transcription activator‐like effector nucleases, zinc finger nucleases, and miRNA‐regulated clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). Genome editing systems have helped in the transformation of metabolic engineering, allowing researchers to modify biosynthetic pathways of different secondary metabolites. Given the growing relevance of editing genomes in plant research, the exciting novel methods are briefly reviewed in this chapter. Also, this chapter highlights recent discoveries on the CRISPR‐based modification of natural products in different medicinal plants.
... Software programs like "DNA works" (Hoover 2012) are efficient in designing TALE domains by accurately calculating oligonucleotides used in amplifying whole gene for oligonucleotide assembly in two-step PCR. Numerous schemes of modular assembly for engineering TALEN constructs are reported (Cermak et al. 2011;Morbitzer et al. 2011;Geissler et al. 2011;Weber et al. 2011). All the systematic approaches for engineering DNA binding domains are also coherent in designing respective domains for ZFN. ...
Currently, soybean (Glycine max (L.) Merrill), a Leguminosae family member, has become one of the main economical oilseed beans. It is being cultivated nowadays in all major areas of the world including China, Japan, Brazil, the USA, and Korea as well as in many South and Midwest countries for several uses. The reason lies in the introduction of multiple local varieties, efficient seed supply, and timely technology transfer, participation of the public sector as well as large international capital groups, and large-scale introduction of new soy foods. It is primarily being cultivated as a substitute for high-protein meat and a source of vegetable oil. Furthermore, the availability of many bioactive compounds has also increased the interest of various researchers toward this bean which originated from northeast China. As a result, it has emerged somewhere as one of the nutritious cum economical parts of the vegan diet. Due to its nutritive value, this “yellow meat of the field” is touted by many as a potential weapon against global hunger. Next to diet, soybean and related greater market value products are being employed either directly or as an ingredient in making cheese, spreads, paints, fertilizers, adhesives, fire extinguisher fluids, animal feed, etc. Due to all these applications, soybean was cultivated on nearly 125 million hectares of the area resulting in 348.7 million tons of harvest in the year 2018. This quantity of production is projected to increase in the near future with a parallel surge in purchasing demand of the every second increasing population. However, there are still many “yield limiters” that uneven the soybean production at both pilot and global scales by nearly 50%. In order to tackle all these soybean yield limiters in a highly efficient manner, various techniques including cross hybridization, molecular marker-assisted breeding, transgenic breeding, tilling, microbiome engineering, and genome editing are being employed by various research groups. Therefore, in the present chapter, the focus is solely on how with time the soybean has proved its strong candidature as a key player for global food security. Furthermore, the production trends at the world and Indian scale are also highlighted. Additionally, the present chapter is an attempt to provide a streamlined overview of all these soybean yield limiters and employed technologies, in brief, to pave the way for the readers for other chapters in the book.KeywordsSoybeanChinaNutritionYieldYield limiters
... To generate a knockout in the SP3C gene, a CRISPR/Cas9 vector containing two guide RNAs (gRNAs) was designed to direct the Cas9 enzyme to two coding regions in the first and fourth exon. The gRNAs 5ʹ-CAACCTCGCGTCGAAATTGG-3ʹ and 5ʹ-TGGATCCCAATTACCGGCTT-3ʹ were artificially synthesized and cloned into the pMDC32 vector (Curtis and Grossniklaus, 2003) by a Golden Gate reaction (Weber et al., 2011). The plasmids were confirmed by Sanger sequencing and then used for transformation of A. tumefaciens (GV3101 strain) by thermal shock. ...
Allelic variation in the CETS (CENTRORADIALIS, TERMINAL FLOWER 1, SELF PRUNING) gene family controls agronomically important traits in many crops. CETS genes encode phosphatidylethanolamine-binding proteins that have a central role in the timing of flowering as florigenic and anti-florigenic signals. The great expansion of CETS genes in many species suggests that the functions of this family go beyond flowering induction and repression. Here, we characterized the tomato SELF PRUNING 3C (SP3C) gene, and show that besides acting as a flowering repressor it also regulates seed germination and modulates root architecture. We show that loss of SP3C function in CRISPR/Cas9-generated mutant lines increases root length and reduces root side branching relative to the wild type. Higher SP3C expression in transgenic lines promotes the opposite effects in roots, represses seed germination, and also improves tolerance to water stress in seedlings. These discoveries provide new insights into the role of SP paralogs in agronomically relevant traits, and support future exploration of the involvement of CETS genes in abiotic stress responses.
... Thus, in contrast to ZFNs, meganucleases, and TALENs which require sophisticated protein engineering to establish specific target site recognition (Smith et al. 2000(Smith et al. , 2006Arnould et al. 2006;Maeder et al. 2008;Boch et al. 2009;Weber et al. 2011), the ease of programmability, simplicity and cost-effectiveness associated with this class of RNA-guided nucleases have significantly advanced medical and agriculture discovery and applications. To that end, since 2012, a spectrum of human clinical applications have emerged, including successful treatment of sickle cell disease (Frangoul et al. 2020) and the historic transplant of a modified pig heart into a human patient with terminal heart disease (Reardon 2022). ...
Abstract:
The selection and introduction of disease resistance genes in livestock not only provide health benefits to animals but opportunities for breeders and farmers to meet the growing demand for high-quality meat and milk while reducing agriculture’s footprint on the environment. As traditional methods of classical breeding and selection for trait improvement are slow, recent progress in several areas of biology including a) understanding host-pathogen interactions, b) inexpensive and rapid DNA sequencing, and c) robust gene editing like CRISPR-Cas provide geneticists tools to accelerate discovery and deployment of disease resistance alleles in livestock. Using these advances, the introduction of resistance genes into commercially relevant germplasm requires access to genetically superior livestock, an infrastructure for scalable allele deployment, freedom to operate, global regulatory approvals, and acceptance of gene edited livestock by producers and consumers. Importantly, academic researchers have recently discovered that modification of the CD163 gene in pigs can confer resistance to the virus that causes porcine reproductive and respiratory syndrome (PRRS). While this achievement represents a major step towards solving an important disease in livestock, to realize the positive impact on animal health while benefiting the pork industry and consumers, it is necessary to introduce this recessive disease resistance allele into commercial breeding populations. Rather than backcrossing the resistance gene from a few non-commercial founders, as a global supplier of high genetic merit livestock genetics, Genus plc and its pig division PIC (Pig Improvement Company) with Genus R&D have mobilized advances in reproductive biology, gene editing, DNA sequencing, and bioinformatics to simultaneously generate and introduce a single modified CD163 allele across four genetically diverse porcine lines of commercial importance that prevents PRRS virus (PRRSV) infection. This report focuses on technical aspects for a scaled gene editing program to consider for rapid and efficient generation and advancement of a small population of non-transgenic founder pigs for commercial breeding. This high genetic merit herd containing a PRRS disease resistance allele will provide important benefits to animal health and food chain value once approved for commercial sale and export.
