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. Rice SOG1 (OsSOG1) and SOG1-like (OsSGL) were identified as putative AtSOG1 orthologs with complete or partial conservation of the serine-glutamine motifs involved in activation via phosphorylation. In addition to OsSOG1 or OsSGL knockout mutants, OsSOG1 nonphosphorylatable mutants (OsSOG1-7A) were generated by homologous recombination-mediated gene targeting. Based on the analysis of DNA damage susceptibility and the effect on the expression of DNA repair-related genes using these mutants, we have demonstrated that OsSOG1 plays a more important role than OsSGL in controlling DDR and DNA repair. OsSOG1-regulated target genes via CTT (N)7 AAG motifs reported previously as AtSOG1 recognition sites. The loss of transcription activity of OsSOG1-7A was not complete compared with OsSOG1-knockout mutants, raising the possibility that other phosphorylation sites might be involved in, or that phosphorylation might not be always required for, the activation of OsSOG1. Furthermore, our findings have highlighted differences in SOG1-mediated DDR between rice and Arabidopsis, especially regarding the transcriptional induction of meiosis-specific recombination-related genes and the response of cell cycle-related genes, revealing rice-specific DDR mechanisms.

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. Radiotracer 28Mg experiments showed that this mutant, named low-magnesium content 1 (LMGC1), has decreased Mg2+ influx in the root and Mg2+ translocation from root to shoot. Consequently, LMGC1 is sensitive to the low Mg condition and prone to develop chlorosis in the young mature leaf. The MutMap method identified a 7.4-kbp deletion in the LMGC1 genome leading to a loss of two genes. Genome editing using CRISPR-Cas9 further revealed that one of the two lost genes, a gene belonging to the RanBP2-type zinc-finger family that we named RanBP2-TYPE ZINC FINGER1 (OsRZF1), was the causal gene of the low Mg phenotype. OsRZF1 is a nuclear protein and may have a fundamental role in maintaining Mg homeostasis in rice 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. Thereafter, advances in genome-editing technologies using CRISPR/Cas9 developed rapidly, with base editing systems for transition substitution using a combination of Cas9 nickase and either cytidine or adenosine deaminase being reported in 2016 and 2017, respectively, and later in 2021 bringing reports of transversion substitution using Cas9 nickase, cytidine deaminase and uracil DNA glycosylase. Moreover, technologies for gene targeting and prime editing systems using DNA or RNA as donors have also been developed in recent years. Besides these precise genome-editing strategies, reports of successful chromosome engineering using CRISPR/Cas9 have been published recently. The application of genome editing to crop breeding has advanced in parallel with the development of these technologies. Genome-editing enzymes can be introduced into plant cells, and there are now many examples of crop breeding using genome-editing technologies. At present, it is no exaggeration to say that we are now in a position to be able to modify a gene precisely and rearrange genomes and chromosomes in a predicted way. In this review, we introduce and discuss recent highlights in the field of precise gene editing, chromosome engineering and genome engineering technology in plants.

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. This system allows integration into the host genome of piggyBac carrying both CRISPR/Cas9 and positive selection marker expression cassettes from an extrachromosomal double-stranded transfer DNA (dsT-DNA), with subsequent excision of the transgenes by the re-transposition of piggyBac from the host genome after successful induction of targeted mutagenesis via CRISPR/Cas9. Here, we demonstrate that the transgenesis system via piggyBac transposition from T-DNA works to deliver transgenes in rice. Following positive-negative selection to exclude transgenic cells randomly transformed with T-DNA, piggyBac-mediated transgenesis from the extrachromosomal dsT-DNA was successful in ca. 1% of transgenic callus lines. After temporary expression of CRISPR/Cas9 within piggyBac, we confirmed, in a proof-of-concept experiment, that piggyBac could be excised precisely from the genome via the stably transformed transposase PBase. Even after excision of piggyBac, CRISPR/Cas9-induced targeted mutations could be detected in the endogenous gene in regenerated rice plants. These results suggest that our piggyBac-mediated transgenesis system will be a valuable tool in establishing efficient CRISPR/Cas9-mediated targeted mutagenesis in vegetatively propagated crops.

Precision genome editing in plants via gene targeting and subsequent break-induced single-strand annealing.

Genome editing via artificial nucleases such as CRISPR/Cas9 has become popular in plants now. However, small insertions or deletions are major mutations and nucleotide substitutions rarely occur when DNA cleavage is induced. To induce nucleotide substitutions, a base editor utilizing dead or nickase-type Cas9 fused with deaminase have been developed. However, the direction and position of practical substitution are still limited. In this context, homologous recombination (HR)-mediated gene targeting (GT) has advantages because any mutations existing on the donor DNA are copied and passed onto the endogenous DNA. As HR-mediated GT is extremely rare in higher plants, positive-negative selection has been used to isolate cells in which GT has occurred. After successful selection, positive selection marker is no longer needed and should ideally be eliminated. In a previous study, we reported a seamless piggyBac-transposon-mediated marker elimination system. Precision marker elimination efficiency in this system is very high. The piggyBac transposon integrates into the host genome at TTAA elements and excises without leaving a footprint at the excised site, so a TTAA sequence is necessary at the location of a positive selection marker. To compensate for this limitation, we have developed a novel marker elimination system using an I-SceI break and subsequent single-strand annealing (SSA)-mediated DNA repair system.

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. We conducted quantitative transformation assays of wild-type and polQ mutant Arabidopsis and rice, analyzed T-DNA/plant DNA junction sequences, and (for Arabidopsis) measured the amount of integrated T-DNA in mutant and wild-type tissue. Unexpectedly, we were able to generate stable transformants of all tested lines, although the transformation frequency of polQ mutants was c. 20% that of wild-type plants. T-DNA/plant DNA junctions from these transformed rice and Arabidopsis polQ mutants closely resembled those from wild-type plants, indicating that loss of PolQ activity does not alter the characteristics of T-DNA integration events. polQ mutant plants show growth and developmental defects, perhaps explaining previous unsuccessful attempts at their stable transformation. We suggest that either multiple redundant pathways function in T-DNA integration, and/or that integration requires some yet unknown pathway.

Allelic Mutations in the Ripening -Inhibitor Locus Generate Extensive Variation in Tomato Ripening.

RIPENING INHIBITOR (RIN) is a transcription factor with transcriptional activator activity that plays a major role in regulating fruit ripening in tomato (Solanum lycopersicum). Recent studies have revealed that (1) RIN is indispensable for full ripening but not for the induction of ripening; and (2) the rin mutation, which produces nonripening fruits that never turn red or soften, is not a null mutation but instead converts the encoded transcriptional activator into a repressor. Here, we have uncovered aspects of RIN function by characterizing a series of allelic mutations within this locus that were produced by CRISPR/Cas9. Fruits of RIN-knockout plants, which are characterized by partial ripening and low levels of lycopene but never turn fully red, showed excess flesh softening compared to the wild type. The knockout mutant fruits also showed accelerated cell wall degradation, suggesting that, contrary to the conventional view, RIN represses over-ripening in addition to facilitating ripening. A C-terminal domain-truncated RIN protein, encoded by another allele of the RIN locus (rinG2), did not activate transcription but formed transcription factor complexes that bound to target genomic regions in a manner similar to that observed for wild-type RIN protein. Fruits expressing this truncated RIN protein exhibited extended shelf life, but unlike rin fruits, they accumulated lycopene and appeared orange. The diverse ripening properties of the RIN allelic mutants suggest that substantial phenotypic variation can be produced by tuning the activity of a transcription factor.

Potato Virus X Vector-Mediated DNA-Free Genome Editing in Plants.

Genome editing technology is important for plant science and crop breeding. Genome-edited plants prepared using general CRISPR-Cas9 methods usually contain foreign DNA, which is problematic for the production of genome-edited transgene-free plants for vegetative propagation or highly heterozygous hybrid cultivars. Here, we describe a method for highly efficient targeted mutagenesis in Nicotiana benthamiana through the expression of Cas9 and single-guide (sg)RNA using a potato virus X (PVX) vector. Following Agrobacterium-mediated introduction of virus vector cDNA, >60% of shoots regenerated without antibiotic selection carried targeted mutations, while ≤18% of shoots contained T-DNA. The PVX vector was also used to express a base editor consisting of modified Cas9 fused with cytidine deaminase to introduce targeted nucleotide substitution in regenerated shoots. We also report exogenous DNA-free genome editing by mechanical inoculation of virions comprising the PVX vector expressing Cas9. This simple and efficient virus vector-mediated delivery of CRISPR-Cas9 could facilitate transgene-free gene editing in plants.

CYP81A P450s are involved in concomitant cross-resistance to acetolactate synthase and acetyl-CoA carboxylase herbicides in Echinochloa phyllopogon.

Californian populations of Echinochloa phyllopogon have evolved multiple-herbicide resistance (MHR), posing a threat to rice production in California. Previously, we identified two CYP81A cytochrome P450 genes whose overexpression is associated with resistance to acetolactate synthase (ALS) inhibitors from two chemical groups. Resistance mechanisms to other herbicides remain unknown. We analyzed the sensitivity of an MHR line to acetyl-CoA carboxylase (ACCase) inhibitors from three chemical groups, followed by an analysis of herbicide metabolism and segregation of resistance of the progenies in sensitive (S) and MHR lines. ACCase herbicide metabolizing function was investigated in the two previously identified P450s. MHR plants exhibited resistance to all the ACCase inhibitors by enhanced herbicide metabolism. Resistance to the ACCase inhibitors segregated in a 3 : 1 ratio in the F2 generation and completely co-segregated with ALS inhibitor resistance in F6 lines. Expression of the respective P450 genes conferred resistance to the three herbicides in rice, which is in line with the detection of hydroxylated herbicide metabolites in vivo in transformed yeast. CYP81As are super P450s that metabolize multiple herbicides from five chemical classes, and concurrent overexpression of the P450s induces metabolism-based resistance to the three ACCase inhibitors in MHR E. phyllopogon, as it does to ALS inhibitors.

Targeted deletion of rice retrotransposon Tos17 via CRISPR/Cas9.

KEY MESSAGE: A successful example of transposon deletion via CRISPR/Cas9-mediated genome editing suggests a novel alternative approach to plant breeding. Transposition of transposable elements (TEs) can affect adjacent genes, leading to changes in genetic traits. Expression levels and patterns, splicing and epigenetic status, and function of genes located in, or near, the inserted/excised locus can be affected. Artificial modification of loci adjacent to TEs, or TEs themselves, by genome editing could mimic the translocation of TEs that occurs in nature, suggesting that it might be possible to produce novel plants by modification of TEs via genome editing. To our knowledge, there are no reports thus far of modification of TEs by genome editing in plants. In this study, we performed targeted deletion of the Tos17 retrotransposon, which is flanked at both ends by long terminal repeat (LTR) sequences, via genome editing in rice. We succeeded in targeted mutagenesis of the LTR, and targeted deletion between LTRs, in calli transformed with CRISPR/Cas9 vectors for the Tos17 LTR. Moreover, we also successfully obtained regenerated plants derived from transformed calli and plants homozygous for lacking Tos17 in the next generation. Taken together, our results demonstrate successful deletion of the Tos17 retrotransposon from the rice genome by targeted mutagenesis using CRISPR/Cas9. We believe that this strategy could be applied to other TEs in many plant species, providing a rapid breeding technology as an alternative means to re-activate expression of agronomically important genes that have been inactivated by TE insertion, especially in plants such as fruit trees, in which it is difficult to maintain parental agronomical traits by cross-breeding due to high heterozygosity.

A removable virus vector suitable for plant genome editing.

Plant genome editing is achieved by the expression of sequence-specific nucleases (SSNs). RNA virus vector-mediated expression of SSNs is a promising approach for transgene integration-free targeted mutagenesis in plants. However, the removal of virus vectors from infected plants is challenging because no antiviral drugs are available against plant viruses. Here, we developed a removable RNA virus vector that carries the target site of tobacco microRNA398 (miR398) whose expression is induced during shoot regeneration. In the inoculated leaves in which expression of miR398 is not induced, insertion of the miR398 target site did not affect the practicability of the virus vector. When shoots were regenerated from the infected leaves, miR398 was expressed and viral RNA was eliminated. The virus vector successfully expressed SSNs in inoculated leaves, from which virus-free genome-edited plants were regenerated via tissue culture.

Simultaneous site-directed mutagenesis of duplicated loci in soybean using a single guide RNA.

KEY MESSAGE: Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T2 seeds. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD). Most of the T1 plants had heterozygous and/or chimeric mutations for the targeted loci. The sequencing analysis of T1 and T2 generations indicates that putative mutation induced in the T0 plant is transmitted to the T1 generation. The inheritable mutation induced in the T1 plant was also detected. This result indicates that continuous induction of mutations during T1 plant development increases the occurrence of mutations in germ cells, which ensures the transmission of mutations to the next generation. Simultaneous site-directed mutagenesis in both GmPPD loci was confirmed in at least 33% of T2 seeds examined. Approximately 19% of double mutants did not contain the Cas9/gRNA expression construct. Double mutants with frameshift mutations in both GmPPD1 and GmPPD2 had dome-shaped trifoliate leaves, extremely twisted pods, and produced few seeds. Taken together, our data indicate that continuous induction of mutations in the whole plant and advancing generations of transgenic plants enable efficient simultaneous site-directed mutagenesis in duplicated loci in soybean.

In Planta Processing of the SpCas9-gRNA Complex.

In CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9)-mediated genome editing in plants, Streptococcus pyogenes Cas9 (SpCas9) protein and the required guide RNA (gRNA) are, in most cases, expressed from a stably integrated transgene. Generally, SpCas9 protein is expressed from an RNA polymerase (pol) II promoter, while gRNA is expressed from a pol III promoter. However, pol III promoters have not been much characterized other than in model plants, making it difficult to select appropriate promoters for specific applications, while pol II transcripts have to be processed to generate functional gRNAs. Recently, successful processing of a pol II transcript into functional gRNAs using ribozyme or Csy4-RNA cleavage systems has been demonstrated. Here, we show that functional gRNAs can be efficiently processed using SpCas9 protein and plant endogenous RNA cleavage systems without the need for a specific RNA processing system. In our system, SpCas9 RNA and gRNA are both transcribed as a single RNA using a single pol II promoter; translated SpCas9 protein can be bound to this RNA and, finally, extra RNA sequences are trimmed by plant RNA processing systems to form a functional SpCas9-gRNA complex. The efficiency of targeted mutagenesis using our novel SpCas9-gRNA fused system was comparable with that of the SpCas9-gRNA system with ribozyme sequence, achieving rates of up to 100% in rice. Our results could be useful in developing stable SpCas9-gRNA expression systems and in RNA virus vector-mediated genome editing systems in plants.

A Split Staphylococcus aureus Cas9 as a Compact Genome-Editing Tool in Plants.

Split-protein methods-where a protein is split into two inactive fragments that must re-assemble to form an active protein-can be used to regulate the activity of a given protein and reduce the size of gene transcription units. Here, we show that a Staphylococcus aureus Cas9 (SaCas9) can be split, and that split-SaCas9 expressed from Agrobacterium can induce targeted mutagenesis in Nicotiana benthamiana. Since SaCas9 is smaller than the more commonly used Cas9 derived from Streptococcus pyogenes, the split-SaCas9 provides the smallest tool yet for clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) plant genome editing. Both sets of split-SaCas9 (_430N/431C and _739N/740C) exhibited genome-editing activity, and the activity of split-SaCas9_739N/740C was almost the same as that of full-length SaCas9. This result indicates that split-SaCas9_739N/740C is suitable for use in targeted mutagenesis. We also show that the split-SaCas9 fragment expressed from Tomato mosaic virus could induce targeted mutagenesis together with another fragment expressed from Agrobacterium, suggesting that a split-SaCas9 system using a plant virus vector is a promising tool for integration-free plant genome editing. Split-SaCas9 has the potential to regulate CRISPR/Cas9-mediated genome editing activity in plant cells both temporally and spatially.

Biallelic Gene Targeting in Rice.

Sequence-specific nucleases (SSNs) have been used successfully in homology-directed repair (HDR)-mediated gene targeting (GT) in many organisms. However, break-induced GT in plants remains challenging due to inefficient delivery of HDR templates and SSNs into plant nuclei. In many plants, including rice, Agrobacterium-mediated transformation is the most practical means of transformation because this biotic transformation system can deliver longer and more intact DNA payloads with less incorporation of fragmented DNA compared with physical transformation systems such as polyethylene glycol, electroporation, or biolistics. Following infection with Agrobacterium, transfer of transfer DNA (T-DNA) to the nucleus and its integration into the plant genome occur consecutively during cocultivation, thus timing the induction of DNA double-strand breaks (DSBs) on the target gene to coincide with the delivery of the HDR template is crucial. To synchronize DSB induction and delivery of the HDR template, we transformed a Cas9 expression construct and GT vector harboring the HDR template with guide RNAs (gRNAs) targeting the rice acetolactate synthase (ALS) gene either separately or sequentially into rice calli. When gRNAs targeting ALS were transcribed transiently from double-stranded T-DNA containing the HDR template, DSBs were induced in the ALS locus by the assembled Cas9/gRNA complex and homologous recombination was stimulated. Contrary to our expectations, there was no great difference in GT efficiency between Cas9-expressing and nonexpressing cells. However, when gRNA targeting DNA ligase 4 was transformed with Cas9 prior to the GT experiment, GT efficiency increased dramatically and more than one line exhibiting biallelic GT at the ALS locus was obtained.

A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice.

We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing.

Overexpression of TIFY genes promotes plant growth in rice through jasmonate signaling.

Because environmental stress can reduce crop growth and yield, the identification of genes that enhance agronomic traits is increasingly important. Previous screening of full-length cDNA overexpressing (FOX) rice lines revealed that OsTIFY11b, one of 20 TIFY proteins in rice, affects plant size, grain weight, and grain size. Therefore, we analyzed the effect of OsTIFY11b and nine other TIFY genes on the growth and yield of corresponding TIFY-FOX lines. Regardless of temperature, grain weight and culm length were enhanced in lines overexpressing TIFY11 subfamily genes, except OsTIFY11e. The TIFY-FOX plants exhibited increased floret number and reduced days to flowering, as well as reduced spikelet fertility, and OsTIFY10b, in particular, enhanced grain yield by minimizing decreases in fertility. We suggest that the enhanced growth of TIFY-transgenic rice is related to regulation of the jasmonate signaling pathway, as in Arabidopsis. Moreover, we discuss the potential application of TIFY overexpression for improving crop yield.

Precision genome editing in plants via gene targeting and piggyBac-mediated marker excision.

Precise genome engineering via homologous recombination (HR)-mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR-mediated GT is an extremely rare event, positive-negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re-integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)-tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.

Precision Targeted Mutagenesis via Cas9 Paired Nickases in Rice.

Recent reports of CRISPR- (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) mediated heritable mutagenesis in plants highlight the need for accuracy of the mutagenesis directed by this system. Off-target mutations are an important issue when considering functional gene analysis, as well as the molecular breeding of crop plants with large genome size, i.e. with many duplicated genes, and where the whole-genome sequence is still lacking. In mammals, off-target mutations can be suppressed by using Cas9 paired nickases together with paired guide RNAs (gRNAs). However, the performance of Cas9 paired nickases has not yet been fully assessed in plants. Here, we analyzed on- and off-target mutation frequency in rice calli and regenerated plants using Cas9 nuclease or Cas9 nickase with paired gRNAs. When Cas9 paired nickases were used, off-target mutations were fully suppressed in rice calli and regenerated plants. However, on-target mutation frequency also decreased compared with that induced by the Cas9 paired nucleases system. Since the gRNA sequence determines specific binding of Cas9 protein-gRNA ribonucleoproteins at the targeted sequence, the on-target mutation frequency of Cas9 paired nickases depends on the design of paired gRNAs. Our results suggest that a combination of gRNAs that can induce mutations at high efficiency with Cas9 nuclease should be used together with Cas9 nickase. Furthermore, we confirmed that a combination of gRNAs containing a one nucleotide (1 nt) mismatch toward the target sequence could not induce mutations when expressed with Cas9 nickase. Our results clearly show the effectiveness of Cas9 paired nickases in delivering on-target specific mutations.

A Universal Positive-Negative Selection System for Gene Targeting in Plants Combining an Antibiotic Resistance Gene and Its Antisense RNA.

Gene targeting (GT) is a useful technology for accurate genome engineering in plants. A reproducible approach based on a positive-negative selection system using hygromycin resistance and the diphtheria toxin A subunit gene as positive and negative selection markers, respectively, is now available. However, to date, this selection system has been applied exclusively in rice (Oryza sativa). To establish a universally applicable positive-negative GT system in plants, we designed a selection system using a combination of neomycin phosphotransferaseII (nptII) and an antisense nptII construct. The concomitant transcription of both sense and antisense nptII suppresses significantly the level of expression of the sense nptII gene, and transgenic calli and plants become sensitive to the antibiotic geneticin. In addition, we were able to utilize the sense nptII gene as a positive selection marker and the antisense nptII construct as a negative selection marker for knockout of the endogenous rice genes Waxy and 33-kD globulin through GT, although negative selection with this system is relatively less efficient compared with diphtheria toxin A subunit. The approach developed here, with some additional improvements, could be applied as a universal selection system for the enrichment of GT cells in several plant species.