An optimised CRISPR Cas9 and Cas12a mutagenesis toolkit for Barley and Wheat.

Background: CRISPR Cas9 and Cas12a are the two most frequently used programmable nucleases reported in plant systems. There is now a wide range of component parts for both which likely have varying degrees of effectiveness and potentially applicability to different species. Our aim was to develop and optimise Cas9 and Cas12a based systems for highly efficient genome editing in the monocotyledons barley and wheat and produce a user-friendly toolbox facilitating simplex and multiplex editing in the cereal community.

Exploring the metabolic and physiological roles of in by gene editing.

The most abundant phenolic compound in Solanaceous plants is chlorogenic acid (CGA), which possesses protective properties such as antimicrobial and antioxidant activities. These properties are particularly relevant when plants are under adverse conditions, such as pathogen attack, excess light, or extreme temperatures that cause oxidative stress. Additionally, CGA has been shown to absorb UV-B light.

Efficient Targeted Mutagenesis in Brassica Crops Using CRISPR/Cas Systems.

CRISPR/Cas has been established for targeted mutagenesis in many plant species since 2013, including Brassica napus and Brassica oleracea. Since that time, improvements have been made in terms of efficiency and choice of CRISPR systems. This protocol encompasses improved Cas9 efficiency and an alternative Cas12a system, allowing more challenging and diverse editing outcomes to be achieved.

Highly Efficient Gene Knockout in Medicago truncatula Genotype R108 Using CRISPR-Cas9 System and an Optimized Agrobacterium Transformation Method.

Medicago truncatula is the model plant species for studying symbioses with nitrogen-fixing rhizobia and arbuscular mycorrhizae, where edited mutants are invaluable for elucidating the contributions of known genes in these processes. Streptococcus pyogenes Cas9 (SpCas9)-based genome editing is a facile means of achieving loss of function, including where multiple gene knockouts are desired in a single generation. We describe how the user can customize our vector to target single or multiple genes, then how the vector is used to make M.

Gene Targeting in Barley Using Cas9 With and Without Geminiviral Replicons.

Advances in the use of RNA-guided Cas9-based genome editing in plants have been rapid over the last few years. A desirable application of genome editing is gene targeting (GT), as it allows a wide range of precise modifications; however, this remains inefficient especially in key crop species. Here, we describe successful, heritable gene targeting in barley at the target site of Cas9 using an strategy but fail to achieve the same using a wheat dwarf virus replicon to increase the copy number of the repair template.

CRISPR-Cas9-Mediated Gene Editing of Genes Impair Glucoraphanin Accumulation of in the Field.

Discoveries in model plants grown under optimal conditions can provide important directions for crop improvement. However, it is important to verify whether results can be translated to crop plants grown in the field. In this study, we sought to study the role of in the regulation of aliphatic glucosinolate (A-GSL) biosynthesis and associated sulfur metabolism in field-grown with the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene-editing technology.

Creating Targeted Gene Knockouts in Brassica oleracea Using CRISPR/Cas9.

While public and political views on genetic modification (inserting "foreign" genes to elicit new traits) have resulted in limited exploitation of the technology in some parts of the world, the new era of genome editing (to edit existing genes to gain new traits/genetic variation) has the potential to change the biotech landscape. Genome editing offers a faster and simpler approach to gene knockout in both single and multiple genetic locations, within a single or small number of generations, in a way that has not been possible through alternative breeding methods. Here we describe an Agrobacterium-mediated delivery approach to deliver Cas9 and dual sgRNAs into 4-day-old cotyledonary petioles of Brassica oleracea.

Creating Targeted Gene Knockouts in Barley Using CRISPR/Cas9.

Knockout mutants are an invaluable reverse genetics tool which have not been well developed in crop species compared to models like Arabidopsis. However, the emergence of CRISPR/Cas9 has changed this situation making the generation of such mutants accessible to many crops including barley. A single T-DNA construct can be transformed into barley immature embryos and stable transgenic lines regenerated through tissue culture which contain targeted mutations.

Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease.

Background: The RNA-guided Cas9 system represents a flexible approach for genome editing in plants. This method can create specific mutations that knock-out or alter target gene function. It provides a valuable tool for plant research and offers opportunities for crop improvement.

Brassica rapa.

Within this chapter we outline an A. tumefaciens-mediated transformation method for B. rapa using 4-day-old cotyledonary explants and the genotype R-o-18.

The same regulatory point mutation changed seed-dispersal structures in evolution and domestication.

It is unclear whether gene regulatory changes that drive evolution at the population and species levels [1-3] can be extrapolated to higher taxonomic levels. Here, we investigated the role of cis-regulatory changes in fruit evolution within the Brassicaceae family. REPLUMLESS (RPL, At5g02030) controls development of the replum, a structure with an important role in fruit opening and seed dispersal [6].

Gibberellins control fruit patterning in Arabidopsis thaliana.

The Arabidopsis basic helix-loop-helix (bHLH) proteins INDEHISCENT (IND) and ALCATRAZ (ALC) specify tissues required for fruit opening that have major roles in seed dispersal and plant domestication. Here, we show that synthesis of the phytohormone gibberellin is a direct and necessary target of IND, and that ALC interacts directly with DELLA repressors, which antagonize ALC function but are destabilized by gibberellin. Thus, the gibberellin/DELLA pathway has a key role in patterning the Arabidopsis fruit, and the interaction between DELLA and bHLH proteins, previously shown to connect gibberellin and light responses, is a versatile regulatory module also used in tissue patterning.

AHP2 is required for bivalent formation and for segregation of homologous chromosomes in Arabidopsis meiosis.

A new Arabidopsis meiotic mutant has been isolated. Homozygous ahp2-1 (Arabidopsis homologue pairing 2) plants were sterile because of failure of both male and female gametophyte development. Fluorescent in situ hybridisation showed that in ahp2-1 male meiocytes, chromosomes did not form bivalents during prophase I and instead seemed to associate indiscriminately.