Plant Science

CRISPR/Cas9 system directed by a gene-specific single guide RNA (sgRNA) is an effective tool for genome editing such as deletions of few bases in coding genes. However, targeted deletion of larger regions generate loss-of-function alleles that offer a straightforward starting point for functional dissections of genomic loci. We present an easy-to-use strategy including a fast cloning dual-sgRNA vector linked to efficient isolation of heritable Cas9-free genomic deletions to rapidly and cost-effectively generate a targeted heritable genome deletion. This step-by-step protocol includes gRNA design, cloning strategy and mutation detection for Arabidopsis and may be adapted for other plant species.

Anabaena sp. PCC 7120 (hereafter Anabaena) is a model cyanobacterium to study nitrogen fixation, cellular differentiation and several other key biological functions that are analogous in plants. As with any other organism, many genes in Anabaena encode an essential life function and hence cannot be deleted, causing a bottleneck in the elucidation of its genomic function. Antisense RNA (asRNA) mediated approach renders the study of essential genes possible by suppressing (but not completely eliminating) expression of the target gene, thus allowing them to function to some extent. Recently, we have successfully implemented this approach using the strong endogenous promoter of the psbA1 gene (D1 subunit of Photosystem II) introduced into a high-copy replicative plasmid (pAM1956) to suppress the transcript level of the target gene alr0277 (encoding a sigma factor, SigJ/Alr0277) in Anabaena. This protocol represents an efficient and easy procedure to further explore the functional genomics, expanding the scope of basic and applied research in these ecologically important cyanobacteria.

CRISPR/Cas9 made targeted mutagenesis and genome editing possible for many plant species. One of the ways that the endonuclease is used for plant genetics is the creation of loss-of-function mutants, which typically result from erroneous DNA repair through non-homologous end joining (NHEJ) pathway. The majority of erroneous repair events results in single-bp insertion or deletion. While single-bp insertions or deletions (indels) effectively destroy the function of protein-coding genes through frameshift, detection is difficult due to the small size shift. High-resolution melting temperature analysis allows quick detection, and it does not require any additional pipetting steps after the PCR amplification of the region of interest. In this protocol, we will describe the steps required for the analysis of potential homozygous mutants.

Chlamydomonas reinhardtii is frequently used as a model organism to study fundamental processes in photosynthesis, metabolism, and flagellar biology. Versatile tool boxes have been developed for this alga (Fuhrmann et al., 1999; Schroda et al., 2000; Schroda, 2006). Among them, forward genetic approach has been intensively used, mostly because of the high efficiency in the generation of hundreds of thousands of mutants by random insertional mutagenesis and the haploid nature therefore phenotypic analysis can be done in the first generation (Cagnon et al., 2013; Tunçay et al., 2013). A major bottleneck in the application of high throughput methods in a forward genetic approach is the identification of the genetic lesion(s) responsible for the observed phenotype. In this protocol, we describe in detail an improved version of the restriction enzyme site-directed amplification PCR (RESDA-PCR) originally reported in (González-Ballester et al., 2005). The improvement includes optimization of primer combination, the choice of DNA polymerase, optimization of PCR cycle parameters, and application of direct sequencing of the PCR products. These modifications make it easier to get specific PCR products as well as speeding up subcloning steps to obtain sequencing data faster.

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein9 (Cas9) is a simple and efficient tool for genome editing in many organisms including plant and crop species. The sgRNAs of the CRISPR/Cas9 system are typically expressed from RNA polymerase III promoters, such as U6 and U3. In many transformation events, more nucleotides will increase the difficulties in plasmid construction and the risk of wrong integration in genome such as base-pair or fragment missing (Gheysen et al., 1990). And also, in many organisms, Pol III promoters have not been well characterized, and heterologous Pol III promoters often perform poorly (Sun et al., 2015). Thus, we have developed a method using single transcriptional unit (STU) CRISPR-Cas9 system to drive the expression of both Cas9 and sgRNAs from a single RNA polymerase II promoter to achieve effective genome editing in plants.

The CRISPR/Cas9 system has emerged as a powerful tool for gene editing in plants and beyond. We have developed a plant vector system for targeted Cas9-dependent mutagenesis of genes in up to two different target sites in Arabidopsis thaliana. This protocol describes a simple 1-week cloning procedure for a single T-DNA vector containing the genes for Cas9 and sgRNAs, as well as the detection of induced mutations in planta. The procedure can likely be adapted for other transformable plant species.

RNA-guided endonucleases (RGENs) have been used for genome editing in various organisms. Here, we demonstrate a simple method for performing targeted mutagenesis and genotyping in a model moss species, Physcomitrella patens, using RGENs. We also performed targeted mutagenesis in a non-model moss, Scopelophilla cataractae, using a similar method (Nomura et al., 2016), indicating that this experimental system could be applied to a wide range of mosses species.

Dissecting the gene regulatory networks (GRNs) underlying developmental processes is a central goal in biology. The characterization of the GRNs underlying flower development has received considerable attention, however, novel approaches are required to reveal temporal and spatial aspects of these GRNs. Here, we provide an overview of the options available to perform dynamic gene perturbations to identify downstream response genes at specific stages of development in the flowers of Arabidopsis thaliana.