CRISPR AND Plant

BACKGROUND: The green peach aphid (Myzus persicae) is a severe agricultural crop pest that has developed resistance to most current control methods, requiring the urgent development of novel strategies. Plant proteinase inhibitors (PINs) are small proteins that protect plants against pathogens and/or herbivores, likely by preventing efficient protein digestion. RESULTS: We identified 67 protease genes in the transcriptomes of three M. persicae lineages (USDA-Red, G002, and G006)...

Plant defense responses to the soil-borne fungus Verticillium longisporum causing stem stripe disease on oilseed rape (Brassica napus) are poorly understood. In this study, a population of recombinant inbred lines (RILs) using the Arabidopsis thaliana accessions Sei-0 and Can-0 was established. Composite interval mapping, transcriptome data and T-DNA mutant screening identified the NITRATE/PEPTIDE TRANSPORTER FAMILY 5.12 gene (AtNPF5.12) as being associated with disease susceptibility in Can-0. Coimmunoprecipitation revealed interaction between AtNPF5...

The Q transcription factor plays important roles in improving multiple wheat domestication traits such as spike architecture, threshability and rachis fragility. However, whether and how it regulates abiotic stress adaptation remain unclear. We found that the transcriptional expression of Q can be induced by NaCl and abscisic acid treatments. Using the q mutants generated by CRISPR/Cas9 and Q overexpression transgenic lines, we showed that the domesticated Q gene causes a penalty in wheat salt tolerance. Then, we demonstrated that Q directly represses the transcription of TaSOS1-3B and reactive oxygen species (ROS) scavenging genes to regulate Na+ and ROS homeostasis in wheat...

Crop improvement by genome editing involves the targeted alteration of genes to improve plant traits, such as stress tolerance, disease resistance or nutritional content. Techniques for the targeted modification of genomes have evolved from generating random mutations to precise base substitutions, followed by insertions, substitutions and deletions of small DNA fragments, and are finally starting to achieve precision manipulation of large DNA segments. Recent developments in base editing, prime editing and other CRISPR-associated systems have laid a solid technological foundation to enable plant basic research and precise molecular breeding...

The CRISPR/Cas9 gene editing technology has proven to be valuable in crop breeding applications. Understanding and mastering this technology will provide a strong foundation for students majoring in biology, agronomy, and related fields to engage in scientific research and work. To incorporate CRISPR/Cas9 technology into experimental teaching courses at colleges, an innovative teaching experiment entitled "Enhancing the resistance of rice plants to bacterial blight disease using CRISPR/Cas9 technology" was designed...

The CRISPR/Cas9 gene editing system is a versatile technology for modifying gene, playing a crucial role in the study of functional genes and genetic breeding of plants, animals, fungi, and microorganisms. This review provides a comprehensive analysis of the application of this technology in gene research and genetic breeding of edible fungi. The review covers various aspects, including the delivery and expression strategies of Cas9 and sgRNA, genetic transformation methods, mutant screening, and repair strategies for target sites following DNA double-strand breaks...

Xanthomonas oryzae pv. oryzae (Xoo) injects major transcription activator-like effectors (TALEs) into plant cells to activate susceptibility (S) genes for promoting bacterial leaf blight in rice. Numerous resistance (R) genes have been used to construct differential cultivars of rice to identify races of Xoo, but the S genes were rarely considered. Different edited lines of rice cv. Kitaake were constructed using CRISPR/Cas9 gene-editing, including single, double and triple edits in the effector-binding elements (EBEs) located in the promoters of rice S genes OsSWEET11a, OsSWEET13 and OsSWEET14...

Modern genome editing tools particularly CRISPR/Cas9 have revolutionized plant genome manipulation for engineering resilience against changing climatic conditions, disease infestation, as well as functional genomic studies. CRISPR-mediated genome editing allows for editing at a single as well as multiple locations in the genome simultaneously, making it an effective tool for polyploid species too. However, still, its applications are limited to the model crops only. Extending it to crop plants will help improve field crops against the changing climates more rapidly and precisely...

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas 9 (CRISPR-associated protein 9) is a robust DNA-encoded, RNA-mediated sequence-specific nuclease system widely used for genome editing of various plants. Although there are many reports on the assembly of gRNAs and plant transformation, there is no single resource for the complete gene editing methodology in tomato. This chapter provides a comprehensive protocol for designing gRNAs, their assembly into the vector, plant transformation, and final mutant analysis in tomato...

This protocol outlines the construction of a plant transformation plasmid to express both the Cas9 nuclease and individual guide RNA (gRNA), facilitating the induction of double-stranded breaks (DSBs) in DNA and subsequent imprecise repair via the non-homologous end-joining (NHEJ) pathway. The gRNA expression cassettes are assembled from three components. First, the Medicago truncatula U6.6 (MtU6) promoter (352 bp) and scaffold (83 bp) sequences are amplified from a pUC-based plasmid. Additionally, a third fragment, corresponding to the target sequence, is synthesized as an oligonucleotide...

CRISPR/Cas9 stands as a revolutionary and versatile gene editing technology. At its core, the Cas9 DNA endonuclease is guided with precision by a specifically designed single-guide RNA (gRNA). This guidance system facilitates the introduction of double-stranded breaks (DSBs) within the DNA. Subsequent imprecise repairs, mainly through the non-homologous end-joining (NHEJ) pathway, yield insertions or deletions, resulting in frameshift mutations. These mutations are instrumental in achieving the successful knockout of the target gene...

Epigenetic editing, also known as EpiEdit, offers an exciting way to control gene expression without altering the DNA sequence. In this study, we evaluate the application of EpiEdit to plant promoters, specifically the MLO (mildew locus o) gene promoter. We use a modified CRISPR-(d)Cas9 system, in which the nuclease-deficient Cas9 (dCas9) is fused to an epigenetic modifier, to experimentally demonstrate the utility of this tool for optimizing epigenetic engineering of a plant promoter prior to in vivo plant epigenome editing...

Tissue culture optimization protocols limit indica rice breeding. Such a challenge is vital because emergent techniques still rely on tissue culture methods and could allow the breeding of new varieties with higher production and toleration of adverse environmental effects caused by climate change. Genome editing technology, using CRISPR/Cas9, is a fast and precise method for accelerated plant breeding. It limited its use in indica subspecies because of the recalcitrant response to in vitro culture methods...

The Coffea spp. plant is a significant crop in Latin America, Africa, and Asia, and recent advances in genomics and transcriptomics have opened possibilities for studying candidate genes and introducing new desirable traits through genetic engineering. While stable transformation of coffee plants has been reported using various techniques, it is a time-consuming and laborious process. To overcome this, transient transformation methods have been developed, which avoid the limitations of stable transformation...

Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C...

Amylose biosynthesis is strictly associated with granule-bound starch synthase I (GBSSI) encoded by the Waxy gene. Mutagenesis of single bases in the Waxy gene, which induced by CRISPR/Cas9 genome editing, caused absence of intact GBSSI protein in grain of the edited line. The amylose and amylopectin contents of waxy mutants were zero and 31.73%, while those in the wild type were 33.50% and 39.00%, respectively. The absence of GBSSI protein led to increase in soluble sugar content to 37.30% compared with only 10...

BACKGROUND: PIN-FORMED genes (PINs) are crucial in plant development as they determine the directionality of auxin flow. They are present in almost all land plants and even in green algae. However, their role in fern development has not yet been determined. This study aims to investigate the function of CrPINMa in the quasi-model water fern Ceratopteris richardii. RESULTS: CrPINMa possessed a long central hydrophilic loop and characteristic motifs within it, which indicated that it belonged to the canonical rather than the non-canonical PINs...

In tomato (Solanum lycopersicum), the ripening of fruit is regulated by the selective expression of ripening-related genes, and this procedure is controlled by transcription factors (TFs). In the various plant-specific TF families, the no apical meristem (NAM), Arabidopsis thaliana activating factor 1/2 (ATAF1/2), and cup-shaped cotyledon 2 (CUC2; NAC) TF family stands out and plays a significant function in plant physiological activities, such as fruit ripening (FR). Despite the numerous genes of NAC found in the tomato genome, limited information is available on the effects of NAC members on FR, and there is also a lack of studies on their target genes...

A tool for precise, target-specific, efficient and affordable genome editing, it is a dream for many researchers, from those who do basic research to those who use it for applied research. Since 2012, we have the tool that almost fulfils such requirements; it is based on CRISPR/Cas systems. However, even CRISPR/Cas has limitations and obstacles that might surprise its users. In this review, we focus on the most frequently used variant, CRISPR/Cas9 from Streptococcus pyogenes, and highlight the key factors affecting its mutagenesis outcomes...

The main bottleneck in the application of biotechnological breeding methods to woody species is due to the in vitro regeneration recalcitrance shown by several genotypes. On the other side, woody species, especially grapevine (Vitis vinifera L.), use most of the pesticides and other expensive inputs in agriculture, making the development of efficient approaches of genetic improvement absolutely urgent. Genome editing is an extremely promising technique particularly for wine grape genotypes, as it allows to modify the desired gene in a single step, preserving all the quality traits selected and appreciated in elite varieties...