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pKAMA-ITACHI Vectors for Highly Efficient CRISPR/Cas9-Mediated Gene Knockout in Arabidopsis thaliana

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is widely used as a tool for genome engineering in various organisms. A complex consisting of Cas9 and single guide RNA (sgRNA) induces a DNA double-strand break in a sequence-specific manner, resulting in knockout. Some binary vectors for CRISPR/Cas9 in plants have been reported, but there is a problem with low efficiency. Here, we present a newly developed, highly efficient CRISPR/Cas9 vector for Arabidopsis thaliana, pKAMA-ITACHI Red (pKIR), harboring the RIBOSOMAL PROTEIN S5 A (RPS5A) promoter to drive Cas9. The RPS5A promoter maintains high constitutive expression at all developmental stages starting from the egg cell and including meristematic cells. Even in the T1 generation, pKIR induced null phenotypes in some genes: PHYTOENE DESATURASE 3 (PDS3), AGAMOUS (AG) and DUO POLLEN 1 (DUO1). Mutations induced by pKIR were carried in the germ cell line of the T1 generation. Surprisingly, in some lines, 100% of the T2 plants had the adh1 (ALCOHOL DEHYDROGENASE 1) null phenotype, indicating that pKIR strongly induced heritable mutations. Cas9-free T2 mutant plants were obtained by removing T2 seeds expressing a fluorescent marker in pKIR. Our results suggest that the pKIR system is a powerful molecular tool for genome engineering in Arabidopsis.


Introduction
Genome engineering has been rapidly developing and makes it possible to perform specific modification of a target gene. Zinc finger nuclease (ZFN) (Urnov et al. 2010) and transcription activator-like effector nuclease (TALEN) (Cermak et al. 2011) were developed as tools for genome engineering. Both consist of artificial DNA-binding proteins fused to FokI endonuclease. The DNA-binding domain designed to recognize the target DNA sequence binds to the target sequence, and FokI endonuclease cleaves the flanking DNA site. The DNA is cleaved as a doublestrand break (DSB), which triggers the DNA repair machinery, generally resulting in a short insertion or deletion via non-homologous end-joining (NHEJ). Since a frameshift in the open reading frame caused by NHEJ disrupts the target gene, a knockout mutant is obtained. ZFN and TALEN have been widely used for genome engineering in various organisms. However, vector construction is not simple because the DNA-binding domain must be customized and tandemly arrayed via several processes to bind to its target DNA sequence. The nucleotide sequences reported herein have been submitted to DDBJ under accession numbers LC189561 (p35S-Cas9), LC189562 (pX2-Cas9), LC189563 (pKIR1.0), LC189564 (pKIR1.1), LC189565 (pKI1.1) and LC189566 (pKI1.1R). Patent: a patent has been applied for on the method to drive Cas9 using the RPS5A promoter for plant genome editing (patent application number: 2016-171590).
Recently, a novel and revolutionary tool for genome engineering, called CRISPR (clustered regularly interspaced short palindromic repeats), has been developed (Jinek et al. 2012, Cong et al. 2013, Mali et al. 2013. The CRISPR/Cas9 (CRISPR-associated 9) system consists of two components, the Cas9 protein and a single guide RNA (sgRNA). Cas9 loading sgRNA scans along DNA to find a target sequence that matches with the 20 bp of the 5 0 sequence of sgRNA, with a protospacer adjacent motif (PAM) sequence (NGG), and then the Cas9-sgRNA complex induces a DSB at the target site. Since the DSB site is repaired by NHEJ, as is the case in ZFN and TALEN, we can generate knockout plants using CRISPR/Cas9. CRISPR/Cas9 was reported to be successful in producing gene-modified plants in Arabidopsis thaliana (Feng et al. 2013, Mao et al. 2013, Fauser et al. 2014, Feng et al. 2014, Wang et al. 2015, Osakabe et al. 2016, Marchantia polymorpha L. (Sugano et al. 2014), Oryza sativa (Feng et al. 2013, Miao et al. 2013, Solanum lycopersicum (Brooks et al. 2014) and other plant species. However, there remain some unresolved problems. First, there is still room for improvement in the mutation induction efficiency via the floral dip method, which introduces genes to female reproductive tissue, in A. thaliana (Ye et al. 1999). The efficiency in inflorescences, which contain germ cells, is not high in A. thaliana. This results in low efficiency of transmission to the next generation. Secondly, the isolation of plants without T-DNA for the CRISPR/ Cas9 system (referred to as 'Cas9-free') requires a PCR experiment as the use of conventional antibiotic selection would result in the death of plants without the T-DNA.
We developed a novel highly efficient CRISPR/Cas9 vector for A. thaliana. To induce inheritable mutations highly efficiently, we tested promoters for Cas9 expression that are constitutively expressed from an initiating cell (an egg cell or a zygote). In Agrobacterium-mediated transformation by floral dipping of A. thaliana, transgenic plants (T 1 plants) are produced through transformation of the egg cell formed in the dipped inflorescence (Ye et al. 1999). If Cas9 expression and mutations are induced in the initiating cell or at an earlier developmental stage, the mutations are passed to the daughter cells and then all or most of the cells in the plant, including the meristematic region, will have the induced mutation. Indeed, an egg cell-specific EC1 promoter to express Cas9 was recently used to obtain non-mosaic T 1 mutants (Wang et al. 2015). Mosaicism is defined as the presence of cells with different genotypes in a tissue or individual. However, the egg cell-specific Cas9 limits the chance of mutation to a very early embryonic developmental stage. In this study, we used the promoters of RIBOSOMAL PROTEIN S5 A (RPS5A) and WUSCHEL RELATED HOMEOBOX 2 (WOX2). The RPS5A gene is expressed from an early embryonic stage (Weijers et al. 2001) and its promoter is constitutively active, beginning in egg cells (Maruyama et al. 2013). The RPS5A promoter is widely used in imaging analyses to label cells, organelles or structures of interest brightly via expressing fluorescent proteins (Maruyama et al. 2013, Gooh et al. 2015, Liao et al. 2015. Data from imaging analyses show that the RPS5A promoter is capable of supporting high-level gene expression. Continuous Cas9 expression from egg cells achieves mutation induction with high efficiency in the T 1 generation. Furthermore, we attempted to select Cas9-free plants non-destructively using a backbone vector, pFAST-R (Shimada et al. 2010), harboring an expression cassette of OLE1-TagRFP (red fluorescent protein) that exhibits red fluorescence in seeds. As reported recently using a similar seed expression system ), a pFAST system that can reveal the existence of transgenes in seeds would provide us with a simple and powerful way to obtain Cas9-free plants.

Vector construction for the CRISPR/Cas9 system in Arabidopsis thaliana
We prepared binary vectors based on the pFAST-R vector series (Shimada et al. 2010) using three promoters to drive human codon-optimized Cas9 expression: Cauliflower mosaic virus 35S (35S), WOX2 and RPS5A. The 35S promoter is a well-known constitutively active promoter. According to previous reports, WOX2 mRNA was detected in the egg cell (Haecker et al. 2004), while fluorescent proteins driven by the WOX2 promoter were observed in the zygote (Ueda et al. 2011, Gooh et al. 2015. The RPS5A promoter begins expression in egg cells (Maruyama et al. 2013, Gooh et al. 2015. The vector carrying 35Sp::Cas9 was designated as p35S-Cas9, WOX2p::Cas9 was designated as pX2-Cas9, and RPS5Ap::Cas9 was designated as p5A-Cas9 (Fig. 1).
For sgRNA expression, an sgRNA cassette, composed of the AtU6.26 promoter, sgRNA and the Pol III terminator, was also inserted into pFAST-R vectors via restriction enzyme-based cloning or Gibson assembly (Fig. 1). Since pFAST-R vectors harbor OLE1p::OLE1:TagRFP within their T-DNA, the transformants exhibit red fluorescence in their seeds, which helps in transformant selection.
Cas9 driven by the RPS5A promoter induced a knockout phenotype in inflorescences of the T 1 generation PDS3 knockout. To estimate the knockout efficiency of our vectors, we selected PHYTOENE DESATURASE 3 (PDS3, AT4G14210) as a target gene. The null mutant showed an albino phenotype (Qin et al. 2007), which makes it easy to determine whether both PDS3 alleles are disrupted in the observed tissues. We observed T 1 plants transformed by p35S-Cas9 with sgRNA against PDS3 (p35S-Cas9_PDS3). Pairs of leaves in T 1 plants were likely to be a mosaic of green and white areas. We judged the pairs of totally white leaves as 'albino' in the following observations. In cotyledons, 17.7% of T 1 plants displayed the albino phenotype (Fig. 2B). However, as the plants grew, the albino frequency decreased. In the first true leaves, 4.2% had albino leaves and, in the second, 3.1% (Fig. 2B). In contrast, in pX2-Cas9_PDS3 T 1 plants, 26.1% had albino second leaves, while the percentage was 66.7% in p5A-Cas9_PDS3 plants (Fig. 2B). Among p5A-Cas9_PDS3 T 1 plants, we found three phenotype patterns: green, partially green (partially albino) and completely albino ( Fig. 2A).
Next, we measured the amounts of three pigments in inflorescence cells. If mutations are detected in the inflorescence, there is an increased probability that the mutation will be inherited by the next generation. To quantify PDS3 knockout efficiency, we measured the amounts of Chl a, Chl b and carotenoid in the inflorescences of T 1 plants because the pds3 null mutant has impaired biosynthesis of Chl and carotenoid (Qin et al. 2007). GA 3 biosynthesis is disrupted in the pds3 mutant, causing a dwarf phenotype. We collected inflorescences from plants grown on medium with exogenous GA 3 , which partially rescues the pds3 dwarf phenotype and promotes bolting. All Col-0 (n = 13) and p35S-Cas9_PDS3 (n = 12) plants bolted in this medium. Among the 23 observed pX2-Cas9_PDS3 plants, 20 bolted (87%) and, among the 57 observed p5A-Cas9_PDS3 plants, 44 bolted (77%). Compared with the inflorescences of Col-0 and p35S-Cas9_PDS3, those of pX2-Cas9_PDS3 and p5A-Cas9_PDS3 contained lower amounts of pigment ( Fig. 2C). For example, the median amount of Chl a per 1 mg of inflorescence was 730 ng in Col-0, 650 ng in p35S-Cas9, 330 ng in pX2-Cas9_PDS3 and 6.4 ng in p5A-Cas9_PDS3. These results indicate that p5A-Cas9_PDS3 induces mutations more efficiently in both rosette leaves and inflorescences. We designated the p5A-Cas9 vector as pKAMA-ITACHI Red 1.0 (pKIR1.0; Kama-itachi is a mink-like trio of ghosts in Japanese folklore: the first one trips up a target person, the second one injures the person with its nails and the third applies ointment, which is similar to the CRISPR system in terms of scanning, cutting and repairing the target DNA sequence). All subsequent experiments were performed using pKIR.
AGAMOUS knockout. To be transmitted to the next generation, the mutation needs to be harbored in germ cells within the floral meristem. Thus, we examined the knockout level in the floral meristem. We chose AGAMOUS (AG, AT4G18960), which is the gene encoding a transcription factor involved in flower development, as the target gene, and the null mutant has a phenotype of a flower within a flower, called 'double flower' (Yanofsky et al. 1990). If the phenotype is observed in pKIR1.0_AG T 1 plants, it indicates that both AG alleles in the floral meristem are disrupted. We observed 13 T 1 plants, and 12 displayed the double flower phenotype, with one dwarf plant arrested before flowering. Furthermore, in all 12 plants, the phenotype was observed in every flower (Fig. 3). This suggests that both AG alleles in the cells of all floral meristems were mutated by pKIR1.0_AG. pKIR efficiently induced transmittable mutations DUO1 knockout. Next, we estimated the knockout efficiency of pKIR1.0 in a male germ cell (sperm cell). If mutations are detected in sperm cells, then mutations may be inherited by the next generation. DUO1 (DUO POLLEN 1, AT3G60460) encodes a male germline-specific R2R3 MYB transcription factor. In the duo1 null pollen grain, cell division of the generative cell is impaired, resulting in a single non-fertile sperm-like cell (Rotman et al. 2005). We prepared pKIR1.0_DUO1 plants in a qrt1-2 mutant background. Since qrt1-2 has a tetrad pollen phenotype (Francis et al. 2006), it helps us to confirm the zygosity of the pollen mother cell. In T 1 plants, we found five patterns for the duo1 phenotype in pollen tetrads by 4 0 ,6-diamidino-2-phenylindole (DAPI) staining ( Fig. 4A-E). A tetrad displaying two sperm cells in each pollen grain is written as [normal, duo1] = [4, 0]. For example, a tetrad written as [2, 2] displays two pollen grains with two sperm cells and two pollen grains with a single sperm-like cell. A tetrad with a single spermlike cell is written as [0, 4], and the mother cell is inferred to be DUO1 homozygous or to have biallelic mutations. In T 1 plants, we collected three flowers from the first inflorescence and examined the frequency of the duo1 phenotype in the tetrads. There were various frequencies of each phenotype pattern among individuals (Fig. 4F). Some tetrads showed [3, 1] or [1, 3] patterns in T 1 plants. One possibility is that DUO1 in such plants is a weak allele. Another is that the mutation is induced after meiosis in the male germline. However, even in qrt1-2, 8.2% of tetrads showed the [3, 1] pattern (n = 7 flowers). These data indicate that a generative cell infrequently fails to undergo cell division. The [3, 1] and [1, 3] patterns in pKIR1.0_DUO1 T 1 plants may reflect a failure in cell division rather than mutation induction after meiosis.
Notably, plants #7 and #10 had 95.4% and 95.1% [0, 4] tetrads, respectively. Reciprocal crosses of the wild type and these plants showed that plant #7 was slightly fertile (17.2%, n = 3), while #10 was completely infertile (0.0%, n = 4) (Fig. 4G). These data indicate that plant #10 is a duo1 null mutant. Next, we examined the DUO1 sequence of plant #10 using DNA from the first cauline leaf, and found a single T base inserted into the DSB site (Fig. 4H). The cauline leaf of plant #10 was most probably non-mosaic because significantly different peaks for bases were not detected subsequent to the DSB site (Fig. 4H). This result PCR + restriction enzyme or Gibson assembly AtU6.26 promoter sgRNA polyT (pKIR1.0) Fig. 1 CRISPR/Cas9 vectors used in this study. Three promoters, 35S, WOX2 and RPS5A, were used to express Cas9. Backbone vectors were from the pFAST-R series and express the red fluorescent protein TagRFP in transgenic seeds. p35S-Cas9 is based on pFAST-R02, while pX2-Cas9 and pKIR1.0 are based on pFAST-R01. An sgRNA cassette amplified by PCR was inserted into the HindIII or SbfI site by restriction enzyme-based cloning or Gibson assembly. LB, left border; Basta R , basta resistance gene; 35sT, 35S terminator; RB, right border; Hyg R , hygromycin resistance gene; hspT, heat shock protein terminator.
suggests that germ cells, at least in the first inflorescence, were completely null and non-mosaic in DUO1 alleles.
ADH1 knockout. To confirm that mutations by pKIR are efficiently transmitted to the next generation, we examined the transmission efficiency of induced mutations of ADH1 (ALCOHOL DEHYDROGENASE 1, AT1G77120). Since ADH1 encodes an enzyme that converts allyl alcohol into the toxic alcohol, acrolein, wild-type and heterozygous plants are killed and null homozygous or biallelic plants survive in the treatment. We prepared two sgRNAs against different target sequences of ADH1 (pKIR1.0_ADH1-1 and pKIR1.1_ADH1-2; see below for details of pKIR1.1). Dozens of T 2 seeds from independent T 1 plants were treated with allyl alcohol. Seven days after sowing, we measured the survival rate. The median survival rates of pKIR1.0_ADH1-1 and pKIR1.1_ADH1-2 T 2 plants were 43% and 81%, respectively. The rate of Col-0 survival was 0.0% and that of adh1 T-DNA mutants (GABI-Kat 924D03.3) was 96% (Fig. 5A). Surprisingly, we isolated two and five plants in which the T 2 seed survival rate was 100% in the pKIR1.0_ADH1-1 and pKIR1.1_ADH1-2 lines, respectively (Fig. 5A). This phenotype of complete resistance against allyl alcohol indicates that ADH1 alleles were already null in all male and female germ lines in the T 1 generation. To fix an induced mutation in and after the T 2 generation and isolate a Cas9-free plant, the elimination of the CRISPR/Cas9 system, which consists of RPS5Ap::Cas9 and U6.26p::sgRNA in this study, is necessary in T 2 seed selection. Our vector pKIR enabled us to eliminate Cas9 by visualization based on seed fluorescence. Since the T-DNA of pKIR also carries OLE1p::OLE1:TagRFP, which fluoresces red in seeds (Shimada et al. 2010), we could select non-transformants (Cas9-free) or transformants by the absence or presence of red fluorescence, providing an easy and non-destructive strategy for isolating Cas9-free plants. Conventional antibiotic-based selection is unsuited to the isolation of Cas9-free plants because plants without the T-DNA for Cas9 are not able to survive on the selection medium.
We performed Cas9-free plant selection on T 2 seeds of pKIR1.1_ADH1-2 plants that showed the 100% survival phenotype (plants #4, #9, #14, #17 and #24) (Fig. 5A). We selected seeds without red fluorescence (Cas9-free seeds) under a fluorescence stereomicroscope and sowed them after allyl alcohol treatment. Next, we collected their leaves and extracted genomic DNA to sequence ADH1 alleles and confirm the absence of Cas9. In T 2 plants from lines #4, #9 and #17, we found more than two patterns of alleles and, in those from lines #14 and #24, we found a single pattern (Fig. 5B). These data indicate that all germ cells in T 1 plants of lines #14 and #24 had the same mutation. This suggests that the T 1 plants were non-mosaic in ADH1 alleles, which may result from mutation induction in an early embryonic stage in the T 1 generation. None of the T 2 plants harbored the Cas9 expression cassette (Supplementary Fig. S1). Thus, pKIR1.0 enabled the easy and non-destructive isolation of targeted Cas9free mutants in the T 2 generation.

Improvement of pKIR to reduce construction steps
We further improved pKIR1.0 to reduce the number of steps involved in construction. In pKIR1.0 construction, two-step PCR and ligation are required for sgRNA cloning. We developed pKIR1.1, which carries an sgRNA cassette (U6.26p::sgRNA) by default. As shown in Fig. 6, two restriction enzyme sites (AarI) were inserted between the U6.26 promoter and sgRNA scaffold. AarI-digested pKIR1.1 forms four-base overhangs, and a hybridized DNA oligomer with overhangs can be inserted into this site. By annealing two 23 bp primers, we can complete vector construction without PCR. We confirmed that pKIR1.1_PDS3 showed equivalent activity to pKIR1.0_PDS3 ( Supplementary  Fig. S2).

Discussion
The CRISPR/Cas9 system is a powerful genome engineering tool for modifying target DNA sequences in various organisms. Although CRISPR/Cas9 has been widely used in some plant species, including A. thaliana (Luo et al. 2016), increasing its efficiency to isolate knockout mutants of interest will significantly contribute to the study of plants that show genetic redundancy. In this study, we used three promoters (35S, WOX2 and RPS5A) to express Cas9 in A. thaliana, and found that the RPS5A promoter had the highest efficiency (Fig. 2B). In all targeted genes (PDS3, AG, DUO1 and ADH1), Cas9 driven by RPS5A was sufficient to knock out both alleles in meristematic cells of T 1 plants. A binary vector harboring RPS5Ap::Cas9 was constructed and named pKIR, and also contained a TagRFP expression cassette for seeds (OLE1p::OLE1:TagRFP; Shimada et al. 2010). The null phenotype induced by pKIR was observed in germ cells (Fig. 4), and the mutations were efficiently transmitted to the next generation (Fig. 5). Among 24 pKIR1.1_ADH1-2 lines, the median survival rate, denoting an adh1 null rate, was 81%, and five lines showed 100% survival (Fig. 5A).
Various CRISPR/Cas9 systems for A. thaliana have been used for some target genes to examine transmission frequency. For example, Fauser et al. (2014) reported the mutation efficiency of ADH1; the survival rate of T 2 plants was 15.3%. Feng et al. (2014) reported the mutation efficiency of several genes. However, neither report mentioned mosaicism. On the other hand, Wang et al. (2015) reported that non-mosaic T 1 plants were isolated and the average frequency of T 2 plants displaying the mutant phenotype was 24.8%. Although the mutation efficiency depends on the design of the sgRNA and it is difficult to compare the mutation efficiency of different sgRNAs induced by different vectors, our pKIR system produced non-mosaic T 1 plants with high efficiency. The RPS5A promoter, which is used in pKIR for Cas9 expression, is known to begin expression in egg cells (Maruyama et al. 2013) and constitutively maintain expression, even in meristematic cells, which probably contributed to the high efficiency of the mutations reported in this study. In some T 2 lines with the 100% allyl alcohol survival phenotype, we found various patterns of mutations (Fig. 5B). These mutations were probably induced independently in different cell lineages rather than successively in the same lineage, based on the mutation sequences. Moreover, the degree of heterogeneity differed between plant lines (Fig. 5B); some lines had a number of mutation patterns, while others had a single one. The single mutation pattern in T 2 plants could be due to non-mosaic mutation in the zygote stage of T 1 plants. These results suggest that the timing of mutation in apical stem cells was line dependent and occurred anywhere from early developmental stages in zygotes to later developmental stages. Constitutive activity of the RPS5A promoter in meristematic cells may significantly increase the frequency of mutation in stem cells over the entire developmental period.
We used the pFAST system (Shimada et al. 2010) for the easy and non-destructive isolation of Cas9-free plants. Cas9 elimination has two advantages: freedom from off-target mutations and freedom from undesirable mutations of a wild-type allele. With regard to off-target mutations, constitutive Cas9 expression at high levels due to the RPS5A promoter could lead to offtarget mutations in non-targeted genes during and after the T 2 generation. After removal of the T-DNA for the CRISPR/Cas9 system, there is no possibility of off-target mutation. With regard to undesirable mutations, mosaic mutations in heterozygous T 2 mutants can be induced. Mutations within the T-DNA for a rescue experiment to compensate for gene function probably occur as long as the T-DNA for the CRISPR/Cas9 system remains in the genomic DNA. Combination of RPS5Ap::Cas9 and the pFAST system was useful for developing a CRISPR/Cas9 vector.
The efficiency of obtaining mutants of interest depends on various factors. For example, the design of sgRNA significantly influences efficiency (Doench et al. 2014), as shown in the knockout of ADH1 (Fig. 5A). We will continue to improve our pKIR series for better efficiency. For example, to stabilize Cas9 expression and increase the non-mosaic rate, the ADH1 5 0untranslated region may be effective as it is a translational enhancer (Sugio et al. 2008). eSpCas9(1.1), with high specificity for targeted DNA DSBs (Slaymaker et al. 2016), will be useful in reducing off-target mutations. Regarding off-target mutations, a highly efficient CRISPR/Cas9 system may increase the possibility of off-target effects. sgRNA cloning in the pKIR system is easy; the preparation of multiple sgRNAs against a single gene of interest can be done quickly and with little effort. Based on the examination of a few sgRNAs, we avoided mistakenly perceiving the phenotype of an off-target mutation as an on-target mutation (Fig. 5A). A combination of nickase Cas9 and activation-induced cytidine deaminase induces point mutations (Nishida et al. 2016).
The RPS5A promoter was reported to have activity in the division zone of the root meristem (Weijers et al. 2001). This suggests that the pKIR series may induce mutations in the roots of T 1 plants. The RPS5A promoter of A. thaliana was active in Torenia fournieri ovular cells, including egg cells (Susaki et al. 2015), and the U6.26 promoter of A. thaliana was active in Nicotiana benthamiana (Jiang et al. 2013). These reports indicate that the RPS5A and U6.26 promoters do not have species specificity and our vectors could potentially be used in other plant species. Taking into consideration the use of other than pFAST, we prepared a vector harboring both in pENTR/D-TOPO (pKAMA-ITACHI in pENTR/D-TOPO 1.1, called pKI1.1). This vector, pKI1.1, can be applied to any GATEWAY vectors depending on the selection marker (e.g. antibiotics and seed fluorescence). Users can select vectors from the pKI series for their own purposes, as pKI1.1-derived pKI1.1R_PDS3 showed efficiency equivalent to that of pKIR1.0_PDS3 in the knockout of the PDS3 gene ( Supplementary Fig. S3). Fig. 7 summarizes the method to obtain Cas9-free knockout mutants by pKIR1.1. pKIR1.1 also contributes to the rapidity of the procedure, with its simple construction through the design of its sgRNA and two 23 bp primers (Fig. 6). Using the six steps shown in Fig. 7, we can now easily obtain knockout mutants of interest. Our pKI vector series based on RPS5A-driven Cas9 provides a useful tool for genome engineering in plants.

Plant materials and growth conditions
Arabidopsis thaliana accession Columbia (Col-0) was used for the experiments. For the DUO1 sgRNA experiments, the qrt1-2 (ABRC stock name: CS8846) mutant was used. For the ADH1 sgRNA experiments, the adh1 (GABI-Kat line ID: 924D03.3) mutant was used. Seeds sterilized by PPM (Plant Cell Technology) were sown on 0.3% gellan gum plates containing 2.3 g l -1 Murashige and Skoog salts (Duchefa Biochemie), 1% sucrose, 1 Â Gamborg's vitamin solution (Sigma-Aldrich), 0.05% MES and 300 mg l -1 cefotaxime sodium (pH 5.8). After a few days of incubation at 4 C, they were grown at 22 C under continuous light for about 2 weeks and then transferred to soil or rock wool.

Plasmid construction
All primers in this study are listed in Supplementary Table S1. Cas9 (with a FLAG-tag and nuclear localization signal) was amplified by PCR from pX330-U6-Chimeric_BB-CBh-hSpCas9 (a gift from Feng Zhang, Addgene plasmid # 42230, Cong et al. 2013) and cloned into pENTR/D-TOPO (Thermo Fisher Scientific). The product was called Cas9 in pENTR/D-TOPO.
For the 35Sp::Cas9 construct, Cas9 from pENTR/D-TOPO was transferred into pFAST-R02 (Shimada et al. 2010) by LR reaction. For the WOX2p::Cas9 and RPS5Ap::Cas9 constructs, each promoter was amplified by PCR from MU14 (a gift from Minako Ueda) and DKv39 (a gift from Daisuke Kurihara), respectively, using forward primers with NotI and reverse primers with NcoI, then inserted into the NotI and NcoI sites of Cas9 in pENTR/D-TOPO. HspT (heat shock protein 18.2 terminator, Nagaya et al. 2010) was inserted into the AscI site of these vectors by Gibson assembly. The promoters combined with Cas9:HspT in pENTR/D-TOPO were transferred into pFAST-R01 (Shimada et al. 2010) by LR reaction. These products were pX2-Cas9 and pKIR1.0.

Plant transformation and selection of transformants
Binary vectors were introduced into Agrobacterium tumefaciens GV3101 by electroporation. Plant transformation was performed using the floral dip method (Clough and Bent 1998). The transformants that displayed red fluorescence in seeds were picked up by a slightly wet toothpick under a stereomicroscope (model SZX16, Olympus) with X-Cite 120LED (Excelitas Technologies).

Pigment measurement
Plants grown on the above-mentioned medium were transferred onto medium with 1.0 mg l -1 GA 3 to promote bolting. After bolting, the first inflorescence was collected and its weight was measured. To extract pigments, the inflorescence was transferred into 1 ml of N,N-dimethylformamide and kept at 4 C overnight in the dark. The absorbances at 480, 647 and 664 nm were measured using a DS-11 Spectrophotometer (DeNovix Inc.). The amount of pigment was calculated according to Wellburn (1994).

DAPI staining of pollen tetrads
Flowers were dipped into 100 ml of DAPI staining buffer [1 mg ml -1 DAPI, 0.1% Triton X-100, 1 mM EDTA, 0.1 M sodium phosphate (pH 7.0)] following the method of Park et al. (1998). After brief vortexing and centrifugation, the precipitated pollen tetrads were transferred onto a slide glass. The samples were observed using a BX51N microscope (Olympus) and the staining images were captured by an Axiocam 506 camera (Carl Zeiss).

Allyl alcohol treatment of seeds
Seeds for allyl alcohol treatment were collected from T 1 plants with ADH1 sgRNA. The seeds were treated with 20 mM allyl alcohol following the procedure of Jacobs et al. (1988). Sterilization and sowing were performed as described above. To calculate the survival rate, germination was observed 7 d after incubation.

Supplementary data
Supplementary data are available at PCP online.