Precise Base Substitution Using CRISPR/Cas-Mediated Base Editor in Rice.

Base editors, CRISPR/Cas-based precise genome editing tools, enable base conversion at a target site without inducing DNA double-strand breaks. The genome editing targetable range is restricted by the requirement for protospacer adjacent motif (PAM) sequence. Cas9 derived from Streptococcus pyogenes (SpCas9)-most widely used for genome editing in many organisms-requires an NGG sequence adjacent to the target site as a PAM.

Gene Targeting via CRISPR/Cas9-Mediated DNA Double-Strand Break Induction in Rice.

Gene targeting (GT) is a precise genome editing tool to achieve desired modification of a target gene, e.g., introduction of point mutations, knock-in of a reporter gene, or swapping of a functional domain, through homologous recombination.

Agrobacterium-Mediated Transformation and Targeted Mutagenesis Using SpCas12f in Rice.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR-Cas) is an adaptive prokaryote immune system against foreign DNA/RNA that is now applied widely to genome editing. A miniature Cas, CRISPR-Cas12f, is one-half to one-third the size of the CRISPR-Cas9 that is commonly used in genome editing experiments in many organisms, including higher plants. The compactness of CRISPR-Cas12f is expected to be advantageous in terms of vector construction and transformation frequency.

Systemic delivery of engineered compact AsCas12f by a positive-strand RNA virus vector enables highly efficient targeted mutagenesis in plants.

Because virus vectors can spread systemically autonomously, they are powerful vehicles with which to deliver genome-editing tools into plant cells. Indeed, a vector based on a positive-strand RNA virus, potato virus X (PVX), harboring SpCas9 and its single guide RNA (sgRNA), achieved targeted mutagenesis in inoculated leaves of . However, the large size of the gene makes it unstable in the PVX vector, hampering the introduction of mutations in systemic leaves.

Heritable Tissue-Culture-Free Gene Editing in Nicotiana benthamiana through Viral Delivery of SpCas9 and sgRNA.

Conventional plant gene editing requires laborious tissue-culture-mediated transformation, which restricts the range of applicable plant species. In this study, we developed a heritable and tissue-culture-free gene editing method in Nicotiana benthamiana using tobacco ringspot virus (TRSV) as a vector for in planta delivery of Cas9 and single-guide RNA (sgRNA) to shoot apical meristems. Agrobacterium-mediated inoculation of the TRSV vector induced systemic and heritable gene editing in Nicotiana benthamiana PHYTOENE DESATURASE.

Precise genetic engineering with transposon in plants.

Transposons are mobile genetic elements that can move to a different position within a genome or between genomes. They have long been used as a tool for genetic engineering, including transgenesis, insertional mutagenesis, and marker excision, in a variety of organisms. The transposon derived from the cabbage looper moth is one of the most promising transposon tools ever identified because has the advantage that it can transpose without leaving a footprint at the excised site.

Excision of DNA fragments with the system in .

is one of the most popular ornamental plants in the world. However, as is a segmental hexaploid, self-incompatible, and has a sizable heterologous genome, it is difficult to modify its trait systematically. Genome editing technology is one of the attractive methods for modifying traits systematically.

CRISPR/Cas9-mediated disruption of CjACOS5 confers no-pollen formation on sugi trees (Cryptomeria japonica D. Don).

Sugi (Cryptomeria japonica D. Don) is an economically important coniferous tree in Japan. However, abundant sugi pollen grains are dispersed and transported by the wind each spring and cause a severe pollen allergy syndrome (Japanese cedar pollinosis).

Targeted deletion of grape retrotransposon associated with fruit skin color via CRISPR/Cas9 in Vitis labrascana 'Shine Muscat'.

Transposition of transposable elements affect expression levels, splicing and epigenetic status, and function of genes located in, or near, the inserted/excised locus. For example, in grape, presence of the Gret1 retrotransposon in the promoter region of the VvMYBA1a allele at the VvMYBA1 locus suppress the expression of the VvMYBA1 transcription factor gene for the anthocyanin biosynthesis and this transposon insertion is responsible for the green berry skin color of Vitis labrascana, 'Shine Muscat', a major grape cultivar in Japan. To prove that transposons in grape genome can be removed by genome editing, we focused on Gret1 in the VvMYBA1a allele as a target of CRISPR/Cas9 mediated transposon removal.

Genome editing in rice mediated by miniature size Cas nuclease SpCas12f.

Cas9 derived from (SpCas9) is used widely in genome editing using the CRISPR-Cas system due to its high activity, but is a relatively large molecule (1,368 amino acid (a.a.) residues).

Genome editing by introduction of Cas9/sgRNA into plant cells using temperature-controlled atmospheric pressure plasma.

Previously, we developed a technique to introduce a superfolder green fluorescent protein (sGFP) fusion protein directly into plant cells using atmospheric-pressure plasma. In this study, we attempted genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system using this protein introduction technique. As an experimental system to evaluate genome editing, we utilized transgenic reporter plants carrying the reporter genes L-(I-SceI)-UC and sGFP-waxy-HPT.

Mutations in RZF1, a zinc-finger protein, reduce magnesium uptake in roots and translocation to shoots in rice.

Magnesium (Mg) homeostasis is critical for maintaining many biological processes, but little information is available to comprehend the molecular mechanisms regulating Mg concentration in rice (Oryza sativa). To make up for the lack of information, we aimed to identify mutants defective in Mg homeostasis through a forward genetic approach. As a result of the screening of 2,825 M2 seedlings mutated by ion-beam irradiation, we found a rice mutant that showed reduced Mg content in leaves and slightly increased Mg content in roots.

SUPPRESSOR OF GAMMA RESPONSE 1 plays rice-specific roles in DNA damage response and repair.

Land plants are constantly exposed to environmental stresses and have developed complicated defense systems, including DNA damage response (DDR) and DNA repair systems, to protect plant cells. In Arabidopsis (Arabidopsis thaliana), the transcription factor SUPPRESSOR OF GAMMA RESPONSE1 (SOG1) plays a key role in DDR. Here, we focus on DDR in rice (Oryza sativa)-thought to be a simpler system compared with Arabidopsis due to lack of induction of the endocycle even under DNA damage stress.

Genome Editing Technology and Its Application to Metabolic Engineering in Rice.

Genome editing technology can be used for gene engineering in many organisms. A target metabolite can be fortified by the knockout and modification of target genes encoding enzymes involved in catabolic and biosynthesis pathways, respectively, via genome editing technology. Genome editing is also applied to genes encoding proteins other than enzymes, such as chaperones and transporters.

Real-Time Monitoring of Key Gene Products Involved in Rice Photoperiodic Flowering.

Flowering is an important biological process through which plants determine the timing of reproduction. In rice, florigen mRNA is induced more strongly when the day length is shorter than the critical day length through recognition of 30-min differences in the photoperiod. , which encodes a CCT-domain protein unique to monocots, has been identified as a key floral repressor in rice, and , a rice ortholog of the floral activator , is another key floral regulator gene.

Precise Genome Editing in miRNA Target Site via Gene Targeting and Subsequent Single-Strand-Annealing-Mediated Excision of the Marker Gene in Plants.

Gene targeting (GT) enables precise genome modification-e.g., the introduction of base substitutions-using donor DNA as a template.

Enhanced FnCas12a-Mediated Targeted Mutagenesis Using crRNA With Altered Target Length in Rice.

The CRISPR/Cas12a (Cpf1) system utilizes a thymidine-rich protospacer adjacent motif (PAM) and generates DNA ends with a 5' overhang. These properties differ from those of CRISPR/Cas9, making Cas12a an attractive alternative in the CRISPR toolbox. However, genome editing efficiencies of Cas12a orthologs are generally lower than those of SpCas9 and depend on their target sequences.

A Universal System of CRISPR/Cas9-Mediated Gene Targeting Using All-in-One Vector in Plants.

Homologous recombination-mediated genome editing, also called gene targeting (GT), is an essential technique that allows precise modification of a target sequence, including introduction of point mutations, knock-in of a reporter gene, and/or swapping of a functional domain. However, due to its low frequency, it has been difficult to establish GT approaches that can be applied widely to a large number of plant species. We have developed a simple and universal clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated DNA double-strand break (DSB)-induced GT system using an all-in-one vector comprising a CRISPR/Cas9 expression construct, selectable marker, and GT donor template.

Plant genome engineering from lab to field-a Keystone Symposia report.

Facing the challenges of the world's food sources posed by a growing global population and a warming climate will require improvements in plant breeding and technology. Enhancing crop resiliency and yield via genome engineering will undoubtedly be a key part of the solution. The advent of new tools, such as CRIPSR/Cas, has ushered in significant advances in plant genome engineering.

Genome Editing in Commercial Wheat Varieties.

Limitations for the application of genome editing technologies on elite wheat ( L.) varieties are mainly due to the dependency on culture and regeneration capabilities. Recently, we developed an particle bombardment (iPB) method which has increased process efficiency since no culture steps are required to create stably genome-edited wheat plants.

Plant genome editing: ever more precise and wide reaching.

Genome-editing technologies consisting of targeted mutagenesis and gene targeting enable us to modify genes of interest rapidly and precisely. The discovery in 2012 of CRISPR/Cas9 systems and their development as sequence-specific nucleases has brought about a paradigm shift in biology. Initially, CRISPR/Cas9 was applied in targeted mutagenesis to knock out a target gene.

A piggyBac-mediated transgenesis system for the temporary expression of CRISPR/Cas9 in rice.

Targeted mutagenesis via CRISPR/Cas9 is now widely used, not only in model plants but also in agriculturally important crops. However, in vegetative crop propagation, CRISPR/Cas9 expression cassettes cannot be segregated out in the resulting progenies, but must nevertheless be eliminated without leaving unnecessary sequences in the genome. To this end, we designed a piggyBac-mediated transgenesis system for the temporary expression of CRISPR/Cas9 in plants.

Protocol for Genome Editing to Produce Multiple Mutants in Wheat.

Here, we describe a protocol for producing multiple recessive mutants via genome editing in hexaploid wheat () cv. Fielder. Using -delivered CRISPR/Cas9 and three sub-genome-specific primer sets, all possible combinations of single, double, and triple transgene-free mutants can be generated.

Agrobacterium T-DNA integration in somatic cells does not require the activity of DNA polymerase θ.

Integration of Agrobacterium tumefaciens transferred DNA (T-DNA) into the plant genome is the last step required for stable plant genetic transformation. The mechanism of T-DNA integration remains controversial, although scientists have proposed the participation of various nonhomologous end-joining (NHEJ) pathways. Recent evidence suggests that in Arabidopsis, DNA polymerase θ (PolQ) may be a crucial enzyme involved in T-DNA integration.