Accepted Manuscript Title: Efficient targeted mutagenesis in soybean by TALENs and CRISPR/Cas9 Author: Hongyang Du Xuanrui Zeng Meng Zhao Xiaopei Cui Qing Wang Hui Yang Hao Cheng Deyue Yu PII: DOI: Reference:

S0168-1656(15)30178-4 http://dx.doi.org/doi:10.1016/j.jbiotec.2015.11.005 BIOTEC 7298

To appear in:

Journal of Biotechnology

Received date: Revised date: Accepted date:

4-8-2015 5-11-2015 10-11-2015

Please cite this article as: Du, Hongyang, Zeng, Xuanrui, Zhao, Meng, Cui, Xiaopei, Wang, Qing, Yang, Hui, Cheng, Hao, Yu, Deyue, Efficient targeted mutagenesis in soybean by TALENs and CRISPR/Cas9.Journal of Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2015.11.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Efficient targeted mutagenesis in soybean by TALENs and CRISPR/Cas9 Hongyang Du1, Xuanrui Zeng1, Meng Zhao, Xiaopei Cui, Qing Wang, Hui Yang, Hao Cheng* [email protected], Deyue Yu* [email protected] National Center for Soybean Improvement, National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China *Corresponding author. 1

Co-first author

1

Highlights • Both TALENs and Cas9/sgRNA systems can achieve gene targeting in soybean. • The mutation efficiency of TALENs was slightly higher than the Cas9/sgRNA system using the AtU6-26 promoter but much lower than that of Cas9/sgRNA system using the soybean GmU6-16g-1 promoter in hairy roots when targeting PDS. • Cas9/sgRNA system is more suitable for simultaneous editing of multiple homoeoalleles in hairy roots when targeting PDS. • GmU6-16g-1 is a suitable U6 promoter for the expression of sgRNA in soybean.

2

Abstract Gene targeting (GT) is of great significance for advancing basic plant research and crop improvement. Both TALENs (transcription activator-like effectors nucleases) and

CRISPR/Cas9

(clustered

regularly

interspaced

short

palindromic

repeats/CRISPR-associated 9) systems have been developed for genome editing in eukaryotes, including crop plants. In this work, we present the comparative analysis of these two technologies for two soybean genome editing targets, GmPDS11 and GmPDS18. We found GT in soybean hairy roots with a single targeting efficiency range of 17.5% to 21.1% by TALENs, 11.7% to 18.1% by CRISPR/Cas9 using the AtU6-26 promoter, and 43.4% to 48.1% by CRISPR/Cas9 using the GmU6-16g-1 promoter, suggesting that the CRISPR/Cas9 using the GmU6-16g-1 promoter is probably a much more efficient tool compared to the other technologies. Similarly, our double mutation GT efficiency experiment with these three technologies displayed a targeting efficiency of 6.25% by TALENs, 12.5% by CRISPR/Cas9 using the AtU6-26 promoter, and 43.4% to 48.1% by CRISPR/Cas9 using the GmU6-16g-1 promoter, suggesting that CRISPR/Cas9 is still a better choice for simultaneous editing of multiple homoeoalleles. Furthermore, we observed albino and dwarf buds (PDS knock-out) by soybean transformation in cotyledon nodes. Our results demonstrated that both TALENs and CRISPR/Cas9 systems are powerful tools for soybean genome editing.

Keywords: soybean; genome editing; TALENs; CRISPR/Cas9.

3

1. Introduction Targeted genome editing is one of the most sought after technologies for basic plant research and biotechnology (Weinthal et al., 2010). During the infancy of genetic engineering, under a natural state the homologous recombination (HR) rate in multicellular organisms was very low, especially in higher plants. A series of studies have shown that targeted DNA double-strand breaks (DSBs) could trigger gene targeting (GT) through HR-mediated recombination events, thereby positioning DSBs as powerful strategies for the versatile and precise modification of eukaryotic genomes (Hsu et al., 2014). Over the past 25 years, four types of artificial sequence-specific nucleases have been developed to induce DSBs: meganucleases (also named homing endonucleases), zinc finger nucleases (ZFNs), transcription activator-like effectors nucleases (TALENs) (Voytas, 2013), and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) (Hsu et al., 2014; Kumar and Jain, 2015). Both TALENs and CRISPR/Cas9 have been widely used in recent years, in which mutations are introduced by two highly conserved ways, the homologous recombination (HR) and non-homologous end joining (NHEJ). In the former way, the homologous donor DNA is used as a template to restore the DSBs, which often results in gene insertion or replacement. In contrast, NHEJ rejoins the broken ends and frequently introduces small deletions or insertions at the junction of the newly rejoined chromosome (Wyman and Kanaar, 2006). TAL effectors are key virulence factors of plant pathogens that comprised of a central repeat domain composed of 13-28 tandem repeat monomers (Kay et al., 2007). The amino acids of each monomer are highly conserved, except for repeat-variable diresidues (RVDs) at positions 12 and 13. Each monomer recognizes a single base and RVDs dictate the specificity of binding. Breaking the DNA binding code specificity of RVDs makes the decisive breakthrough of this technical application (Boch et al., 2009; Moscou and Bogdanove, 2009). TALENs consist of a customizable “TAL domain” (DNA-binding domain, DBD) and nonspecific endonuclease Fok I cleavage domain, which cleaves as a dimer. In the past few years, TALEN-mediated genome editing has 4

been adopted in various organisms and cells, and a handful of researchers have achieved targeted mutagenesis in plants (Cermak et al., 2011; Li et al., 2012; Mahfouz et al., 2011; Shan et al., 2013a; Zhang et al., 2013). The CRISPR/Cas9 system emerged less than three years ago and quickly became an alternative to the Fok I-based ZFNs and TALENs. CRISPRs are essential components of adaptive immune systems that widely exist in bacteria and archaea that are involved in the degradation of invaded viruses or phage DNA based on a RNA-guiding system (Wiedenheft et al., 2012). The Cas9 endonuclease, a characteristic protein of the type II CRISPR/Cas system (total three types), is a large protein that includes both RuvC-like nuclease domain and HNH nuclease domain. Cas9 forms a complex with two short RNA molecules called trans-activating crRNA (tracrRNA) and CRISPR RNA (crRNA) to guide the nuclease domains in cleaving non-self DNA on both cognate DNA strands that homologous to the spacer. The presence of the protospacer adjacent motif (PAM) 5′-NGG-3′ downstream of the target site is crucial for its cleavage activity. Based on a landmark study, the dual-tracrRNA:crRNA heteroduplex can be replaced by a single chimeric RNA (single guide RNA, sgRNA), thus reprogramming the Cas9/sgRNA system to a specific targeted site (Jinek et al., 2012), subsequently applying in human and mouse cells for the first time in vivo (Cong et al., 2013; Mali et al., 2013). Several labs have demonstrated the effectiveness of the Cas9/sgRNA system in plants (Feng et al., 2013; Jiang et al., 2013; Li et al., 2013; Mao et al., 2013; Miao et al., 2013; Nekrasov et al., 2013; Shan et al., 2013b; Xie and Yang, 2013). Plant genome modifications based on Cas9 are stable and heritable with the capability of recovering homozygous T1 desired lines (Feng et al., 2014; Zhang et al., 2014). Soybean (Glycine max), one of the most important oil and high-protein crops, contains a variety of physiologically active substances that are beneficial to humans. Despite its economic importance, basic genetic and genome editing research of soybean is lagging behind other crops. Targeted mutagenesis of the duplicated fatty acid desaturase 2 genes (FAD2-1A and FAD2-1B) has been achieved in soybean with 5

TALENs, resulting in improved soybean oil quality (Haun et al., 2014). Recently, four studies provided evidence of expression of the Cas9/sgRNA system in soybean. One study targeted mutations in hairy roots and somatic embryos using a Medicago truncatula U6.6 polymerase III promoter for efficient transcription of sgRNA to achieve a mutation efficiency as high as 95% (Jacobs et al., 2015). One study compared the efficiency of the soybean U6-10 and Arabidopsis U6-26 promoter for targeted mutagenesis in hairy roots and protoplasts. The soybean U6-10 promoter showed better efficiency than Arabidopsis U6-26 in driving sgRNA expression (Sun et al., 2015). Another study used Arabidopsis U6-26 promoter for targeted both endogenous and exogenous genes mutagenesis in hairy roots (Cai et al., 2015). The last used Cas9/sgRNA system for targeted gene integrations in soybean embryonic callus and got transmitted T1 generation for analyzing the inheritance across generations (Li et al., 2015). In this paper, we compared the TALENs and CRISPR/Cas9 systems in targeting the gene encoding phytoene desaturase (PDS), a rate-limiting enzyme involved in carotenoid biosynthesis pathway. The functional knock-out mutation of this gene led to an albino and dwarf phenotype, and this phenotype has been observed in rice, a diploid plant which only has one PDS gene copy (Shan et al., 2013b). Our results demonstrated that targeted mutagenesis was successfully achieved through both TALENs and CRISPR/Cas9 systems as further proof of concept in soybean.

2. Material and Methods 2.1. Plasmid construction TALENs were designed by the TAL Effector-Nucleotide Targeter (TALE-NT) 2.0 program (Doyle et al., 2012). For ease of analysis, TALENs recognition sequence contained a restriction enzyme site within the spacer region (Supplemental Table S1). TALENs repeat arrays were constructed by one-step ligation using the FastTALETM TALEN Assembly Kit (SIDANSAI Biotechnology (Shanghai) CO., LTD; Catalog. 6

No.1802-030) and subsequently subcloned into a modified plant transformation binary vector pCAMBIA 1301. For construction of plant expression vectors of Cas9/sgRNA system, we used the Cas9/sgRNA plasmid construction kit (VIEWSOLID Biotechnology (Beijing) CO., LTD; Catalog. No. VK005-04). The plasmid of this kit contained a dicotyledon codon-optimized dpCas9 (under the maize Ubi promoter) and a sgRNA scaffold (under the Arabidopsis AtU6 promoter) (Supplemental Figure 1). Sequences for sgRNAs were identified by the web-based tools: CRISPR-P (Lei et al., 2014) and CRISPR-PLANT (http://www.genome.arizona.edu/crispr/index.html) (Supplemental Table S2). We also constructed a plasmid to replace the AtU6 promoter with a GmU6 promoter cloned from soybean cultivar “Williams 82” (Supplemental Sequence 2). 2.2. Hairy root transformation of soybean Each TALENs and Cas9/sgRNA binary construct was independently transformed into Agrobacterium rhizogenes strain K599 for hairy root transformation. Soybean cotyledons of cultivar “Jack” were inoculated with the transformed K599 strain using a previously described protocol (Liang et al., 2010) with slight modifications. Briefly, seeds were surface sterilized by incubating 6-8 hour in Cl2 gas produced by a mixture of 100 mL of NaClO and 5.0 mL of HCl followed by germination on SG4 medium (½ MS salts, 5 g/L sucrose, 3.5 g/L Phytagel) in a growth room for 5 days at 25°C under a 16 light / 8 dark cycle. The abaxial side of cotyledons was wounded with a scalpel, and the Agrobacterium solution (cultured to an O.D.600 of 0.8) was centrifuged and then suspended in 10 mM MgCl2 buffer with 50 μM acetosyringone to an O.D.600 of 0.5. A drop of suspension was applied on the wounded side of cotyledons and the inoculated cotyledons were grown on White medium containing 500 μg/mL carbenicillin disodium and 50 μg/mL cefotaxime disodium at 25°C in the dark. After two weeks, inoculated cotyledons were transferred to a new medium plate followed by collection of regenerated roots 10 days later. 2.3. Soybean transformation 7

Stable transformation of soybean was carried out using an improved cot-node transformation protocol (Zeng et al., 2004) using Agrobacterium tumefaciens strain EHA105 to transform plasmids. Briefly, seeds of soybean cultivar Jack were surface sterilized and germinated as previously described. Using a scalpel blade, the seed was cut vertically through the hypocotyl region in order to remove the embryonic axis tissue that remained attached to the cotyledon. The cotyledonary-nodes were wounded by making 10 slices perpendicular to the hypocotyl and inoculated in the co-cultivation medium CCM (Liquid: 1/10 B5 salts, 30 g/L sucrose, 20mM MES, 1.67 mg/L BAP, 0.25 mg/L GA3 and 200 μM acetosyringone) for 30 min, and then transferred on co-cultivation medium CCM (Solid: CCM liquid plus 5 g/L agarose, additional 0.4 g/L L-cysteine, 0.154 g/L DTT, 0.158 g/L Na-thiosulfate) for co-cultivation for 5 days in dark condition. The infected explants were briefly washed in Washing Medium (B5 salts, 30 g/L sucrose, 3mM MES, 1.67 mg/L BAP, 500 μg/mL carbenicillin and 50 μg/mL cefotaxime) and transferred to SI Medium (Washing Medium plus 3.5 g/L phytagel). Afterward the explant tissue was cultured for 14 days at 25°C under a 16 h light and 8 h dark cycle. Explant tissue was then transferred to fresh SI medium with 6 mg/L glufosinate for screening. To initiate shoot elongation, explants were transferred to fresh SE medium (MS salt, 30 g/L sucrose, 3.5 g/L phytagel, 3mM MES, 5 mg/L Asparagine, 10 mg/L Pyroglutamic, 0.1 mg/L IAA, 0.5 mg/L GA3, 1 mg/L Zeatin Riboside, 500 μg/mL carbenicillin and 50 μg/mL cefotaxime) with 3 mg/L glufosinate every two weeks. At each transfer a fresh horizontal cut at the base of the tissue was made. To initiate rooting, elongated shoots were placed into rooting medium (MS salt, 20 g/L sucrose, 3.5 g/L phytagel, 3mM MES and 0.5 mg/L IBA). 2.4. Genomic DNA extraction and PCR/sequencing-based genotyping Genomic DNA was isolated from approximately 300 mg of hairy roots samples using the DNAsecure Plant Kit (TIANGEN Biotechnology (Beijing) CO., LTD; Catalog. No. DP320). To identify mutations in targeted genes by PCR, 50 – 100 ng of DNA and Phanta® Max Super-Fidelity DNA Polymerase (Vazyme Biotechnology (Nanjing) 8

CO., LTD; Catalog. No. P505) were used. Primers used were listed in Supplement Table S3. Digestion of PCR products (the spacer has a restriction enzyme site) were performed using a restriction enzyme that recognized the wild-type target sequences. Mutations introduced by NHEJ were resistant to restriction enzyme digestion due to loss of the restriction site and resulted in uncleaved bands. Situations in which no restriction endonuclease was useful for analysis, a mismatch-sensitive T7E1 endonuclease (VIEWSOLID Biotechnology CO., LTD; Catalog. No. E001L) or Cruiser™ enzyme (Genloci Biotechnology CO., LTD; Catalog. No.GP0106) was used to detect mutations. In this approach, PCR products were treated with T7E1/Cruiser™ after melting and annealing, and cleaved DNA fragments were detected if they contained both mutated and wild-type DNA sequences. All enzyme digestion products were visualized by 1.5% agarose gel electrophoresis. Mutated products of interest were then cloned into the TA cloning vector and submitted for sequencing.

3. Results 3.1. TALENs design and activity assessment To evaluate the TALENs technique application in soybean, soybean phytoene desaturase (PDS) genes were targeted. There are two PDS genes in soybean, GmPDS11 (Glyma.11G253000) and GmPDS18 (Glyma.18G003900). We confirmed the sequence of these two genes in the soybean cultivar “Jack”, used as genetic transformation recipient. Sequence-specific TALENs were designed by using TAL Effector-Nucleotide Targeter (TALE-NT) 2.0 program (Doyle et al., 2012). Both GmPDS11 and GmPDS18 are highly homologous paralogs, suggesting they encode enzymes of similar function. We designed two types of TALENs, one that targeted both genes at the homologous sequence region and one that specifically only targeted a single gene. Three TALENs pairs were designed: (1) D1 that targeted both at exon 2 and (2) S1 and S2 that targeted GmPDS11 at exon 4 and 5 respectively (Supplement 9

Table S1). TALENs’ repeat arrays were constructed using the FastTALETM TALEN Assembly Kit (SIDANSAI Biotechnology (Shanghai) CO., LTD). To assess the efficiency of targeted mutagenesis by TALENs in soybean, we used the Agrobacterium rhizogenes hairy-root transformation method, by which transgenic hairy roots can be easily obtained within three weeks to provide an effective means of rapid screening of TALENs function at endogenous targets (Curtin et al., 2011). A total of 217 regenerated roots (Supplemental Figure 2) were inoculated by three TALENs vectors, and genomic DNA was extracted and PCR/restriction enzyme (PCR/RE) assays were carried out to detect mutations (Figure 1). Regenerated hairy roots of sufficient length were taken as independent transformation events. The mutation frequency was calculated as the number of hairy roots showing mutations divided by the total number of hairy roots inoculated by each vector. In this assay, transgenic soybean roots showed mutagenesis frequencies ranging from 17.5% to 21.1% (Table 1), a little higher than the frequencies of mutations in plant protoplasts but a little lower than that in plant calli (Shan et al., 2013a). This may be due to the difference of culture time and inoculated organisms. In our case, most mutations were small deletions ranging from 1 to 30 bp and a few showed inserts (Figure 2), which is consistent with other studies (Li et al., 2012; Shan et al., 2013a; Zhang et al., 2013). Of the root samples transformed with TALENs-D1, 5 out of 80 clones (6.25%) exhibited mutations at both targets (Table 1), suggesting that simultaneous editing of two homoeoalleles in soybean could be achieved, but the efficiency was much lower than that of one target. It has demonstrated that TALENs have a thymine (T) preference at position “0”, but it is not strict (Doyle et al., 2013). In our data, TALENs-S2 had an off-target location on GmPDS18 for cytosine (C) at position “0” of TAL1 (Supplement Table S1). From our study, 5 out of 71 clones (7.0%) exhibited mutations at off-target GmPDS18 and all of them had mutations at GmPDS11, suggesting that TALENs preferentially bind targets that have a “T” at position “0”. 3.2. CRISPR/Cas9 design and activity assessment To evaluate the CRISPR/Cas9 system application in soybean, the same genes were 10

targeted and the same assessment method was used. The CRISPR/Cas9 system requires the Cas9 protein is accompanied with a single guide RNA (sgRNA) that directs the Cas9 endonuclease to a complementary target DNA. Commonly, the sgRNA is under the control of the U3 or U6 snRNA promoter (RNA polymeras III promoter). For our study, a commercial Cas9/sgRNA vector, containing a dicotyledon codon-optimized dpCas9 under the maize Ubi promoter and a sgRNA scaffold under the AtU6-26 promoter were used. Sequence-specific sgRNAs were identified by two web-based tools: CRISPR-P (Lei et al., 2014) and CRISPR-PLANT. A total number of four sgRNAs were designed: (1) D7 that targeted both genes at exon 2; (2) S11 and S12 that targeted GmPDS11 at exon 4 and 6, respectively and (3) S13 that targeted GmPDS18 at exon 5 (Supplement Table S2). Using Arabidopsis U6-26 gene sequence as query, we identified 11 U6 snRNA genes in the soybean genome. One of the snRNA genes was located on chromosome 16 and cloned from cultivar “Williams 82”, hereafter referred to as GmU6-16g-1 (Supplemental Sequence 1). We then constructed a D7 vector in which the AtU6-26 promoter was replaced with the GmU6-16g-1 promoter of different lengths, designated as D7-a (616bp) and D7-b (350bp) (Supplemental Sequence 2). To estimate the efficiency of genome editing, T7 endonuclease I (T7E1) or Cruiser endonuclease assay was performed to detect mutations for all targeted sites using the hairy-root transformation method. In this assay, PCR products were treated with mismatch-sensitive T7E1 or Cruiser after melting and annealing to detect for cleaved DNA fragments if amplified products contained both mutated and wild-type DNA. It should be noted that all detected samples were sequenced. As shown in Figure 1 (C) and (D), T7E1 or Cruiser-digested fragments were detected in the samples but not in the control. Under the control of the AtU6-26 promoter, transgenic soybean roots showed mutagenesis frequencies ranging from 11.7% to 18.1% (Table 2). However, under the control of the soybean GmU6-16g-1 promoter, transgenic soybean roots showed mutagenesis frequencies of 43.4% and 48.1% (Table 3). Differences observed may be due to the higher expression of sgRNA under the soybean U6 promoter. The 11

promoter length for AtU6-26 was 445 bp and for the two different GmU6-16g-1 promoters were 616 bp and 350 bp, respectively. The different genome editing efficiency of the two lengths suggested that the shorter promoter was sufficient for activity (Table 3). Overall, various mutations were found including a large deletion (>20 bp) and several small deletions or insertions (Figure 3). To further our study, the D7 vector was introduced into soybean cotyledon nodes using Agrobacterium tumefaciens. After eight weeks of culture, glufosinate-resistant adventitious buds appeared and total sixteen buds were survived. Those five lines had an obvious albino and dwarf phenotype, and the remaining ones were normal green or slight etiolated (common phenomenon caused by the stress of tissue culture), which were proved to be false positives by strip test (a test that can quickly detect the existence of the PAT, coded by genetic screening gene phosphinothricin acetyltransferase gene (bar)). We here focused on the five albinistic buds. The first two albinistic leaves from two different buds were examined using the strip test (Supplemental Figure 3a and c), and the albinistic leaves of the other three buds were analyzed by Genomic DNA extraction and PCR/sequencing (Figure 4, Supplemental Figure 3b and 4). Those results suggested that the albino and dwarf phenotype shown on buds may be caused by the disruption of PDS, and this phenotype was similar to that observed in rice (Shan et al., 2013b). It must be noted that the albino phenotype did not appear in the beginning of culture, but in a process of the change from green to albino. That means the albino buds we observed may be chimeras, namely the mutations are somatic sectors. In order to get whole-plant mutations and analyze the inheritance across generations, a number of T1/T2 generation plants are required (Feng et al., 2014; Zhang et al., 2014). Considering that a PDS deletion causes a strong negative effect on plant growth, we could not recover a T0 plant but the adventitious buds. However, using cultured buds we demonstrated that Cas9 could be guided by engineered sgRNA for precise cleavage and editing of soybean genome. In summary, we compared the mutation efficiency of the TALENs and Cas9/sgRNA gene targeting systems in soybean hairy roots. The mutation efficiency of TALENs 12

was slightly higher than the Cas9/sgRNA system using the AtU6-26 promoter but much lower than that of Cas9/sgRNA system using the soybean U6 promoter (Table 1, 2 and 3). However, the simultaneous editing of multiple homoeoalleles at homologous sequence, Cas9/sgRNA system using soybean GmU6-16g-1 promoter was much more efficient.

4. Discussion Genome editing using engineered nucleases (GEEN) is an effective genetic engineering method that uses “molecular scissors” to target and digest DNA at specific locations in the genome to induce DSBs that are then repaired by the natural processes of HR or NHEJ. Although TALE DNA binding monomers are for the most part modular, they require context-dependent specificity and the synthesis of novel TALE arrays by PCR can be labor intensive and costly (Voytas, 2013). Unlike the Fok I-based method of TALENs, CRISPR/Cas9 system uses a codon-optimized Cas9 endonuclease and a sgRNA, in which the RNA-guided DNA targeting is facilitated by complementary base pairing. The only constraint is that the recognition sites need to be preceded by a 5′-NGG protospacer-adjacent motif (PAM). Overall, the CRISPR/Cas9 technology is a more straightforward and affordable option for genome editing. Its simplicity makes “gene editing at CRISPR speed” (Baker, 2014). Indeed, genome editing holds significant promise for advancing basic plant research as well as crop improvement (Mahfouz et al. 2014; Voytas and Gao, 2014) considering some successful applications (Li et al., 2012; Wang et al., 2014). In the present study, we tested TALENs and CRISPR/Cas9 systems on soybean by targeting PDS genes. For TALENs, the construction of the vectors was costly and time-consuming and lower mutation efficiencies ranging from 17.5% to 21.1% in soybean hairy root (Table 1), in which most were small deletions or inserts (Figure 2). From this approach, biallelic mutants were detected in hairy root (Figure 1a). Since the early stages of TALENs development, it has been recommended to target a site 13

with a thymine (T) at position “0”. We demonstrated that TALENs could also bind sequences that have a cytosine (C) at position “0”, but the efficiency is lower than that of a thymine (T). The concentration of the Cas9-sgRNA complex is one key factor for the successful editing by the Cas9/sgRNA system (Pattanayak et al., 2013). Therefore, we compared the efficiency of the soybean GmU6-16g-1 and Arabidopsis U6-26 promoters in driving the expression of sgRNA for targeted gene mutagenesis. When using the AtU6-26 promoter, transgenic soybean roots showed mutagenesis frequencies ranging from 11.7% to 18.1%, in contrast to the 43.4% and 48.1% when using the soybean GmU6-16g-1 promoter (Table 2 and 3). We used two different GmU6-16g-1 promoter lengths, and the data showed that the shorter fragment was sufficient for promoter activity. We noticed that the hairy roots incubated by D7-a and D7-b all showed double-mutations. This result may be due to the high efficiency of Cas9/sgRNA system using soybean GmU6-16g-1 promoter. We also noticed that the mutagenesis frequencies of Cas9/sgRNA were higher than that of previous study, in which mutagenesis frequencies were 3.2%–9.7% using the pCas9-AtU6-sgRNA vector and 14.7–20.2% with the pCas9-GmU6-sgRNA vector (Sun et al., 2015). This may be due to the different vector and genetic transformation recipient. Plant genome editing techniques largely depend on plant genetic transformation. In comparison with other crops, soybean transformation efficiency is still low. In the present study we observed five positive buds, and all showed the albino and dwarf phenotype (Figure 4, Supplemental Figure 3).

It seemed that genome editing

frequency of stable genetic transformation was 100%, but this result needs to be further analyzed as the sample size was small (only five samples were used here). PDS deletion has a negative effect on plant growth, thus we could not recover a whole-plant but cultured buds in T0. Sequencing analysis showed that the phenotype may be caused by disruption of PDS, suggesting that genome editing of a whole-plant probably is feasible. A recent article reported that Cas9 induced GT in soybean

14

embryonic callus and T1 progenies that were segregated according to Mendelian laws and clean homozygous T1 plants could be obtained (Li et al., 2015). Off-target effects are crucial for GT techniques’ applications and there have been several studies that focused on solving this issue (Maruyama et al., 2015; Ran et al., 2015; Tsai et al., 2014). Gene knock-outs induced by engineered nucleases is the most widely used application, but the technology is not limited to knock-outs. Taking TALENs and CRISPR/Cas9 systems as genome engineering platforms, we will have broader application prospects (Hsu et al., 2014; Shalem et al., 2015; Sternberg and Doudna, 2015). In conclusion, both TALENs and Cas9/sgRNA systems can achieve GT in soybean. In terms of the simplicity and the cost of the experiment, the Cas9/sgRNA system seems to be a better choice for simultaneous editing of multiple homoeoalleles. In addition, the use of the soybean GmU6-16g-1 promoter showed more effective than the Arabidopsis AtU6-26 promoter for Cas9/sgRNA applications system in soybean.

Acknowledgements This work was supported in part by National Natural Science Foundation of China (31301342, 31370034) and Key Transgenic Breeding Program of China (2014ZX08004-003)

15

References Baker, M., (2014) Gene editing at CRISPR speed. Nat Biotechnol 32, 309-312. Boch, J., Scholze, H., Schornack, S., Landgraf, A., Hahn, S., Kay, S., Lahaye, T., Nickstadt, A., Bonas, U., (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509-1512. Cai, Y., Chen, L., Liu, X., Sun, S., Wu, C., Jiang, B., Han, T., Hou, W., (2015) CRISPR/Cas9-Mediated Genome Editing in Soybean Hairy Roots. PLoS One 10, e0136064. Cermak, T., Doyle, E.L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., Baller, J.A., Somia, N.V., Bogdanove, A.J., Voytas, D.F., (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39, e82. Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., Zhang, F., (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823. Curtin, S.J., Zhang, F., Sander, J.D., Haun, W.J., Starker, C., Baltes, N.J., Reyon, D., Dahlborg, E.J., Goodwin, M.J., Coffman, A.P., Dobbs, D., Joung, J.K., Voytas, D.F., Stupar, R.M., (2011) Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases. Plant Physiol 156, 466-473. Doyle, E.L., Booher, N.J., Standage, D.S., Voytas, D.F., Brendel, V.P., Vandyk, J.K., Bogdanove, A.J., (2012) TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Res 40, W117-122. Doyle, E.L., Hummel, A.W., Demorest, Z.L., Starker, C.G., Voytas, D.F., Bradley, P., Bogdanove, A.J., (2013) TAL Effector Specificity for base 0 of the DNA Target Is Altered in a Complex, Effector- and Assay-Dependent Manner by Substitutions for the Tryptophan in Cryptic Repeat –1. Plos One 8, e82120. Feng, Z., Mao, Y., Xu, N., Zhang, B., Wei, P., Yang, D.L., Wang, Z., Zhang, Z., Zheng, R., Yang, L., Zeng, L., Liu, X., Zhu, J.K., (2014) Multigeneration analysis 16

reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci U S A 111, 4632-4637. Feng, Z., Zhang, B., Ding, W., Liu, X., Yang, D.L., Wei, P., Cao, F., Zhu, S., Zhang, F., Mao, Y., Zhu, J.K., (2013) Efficient genome editing in plants using a CRISPR/Cas system. Cell Res 23, 1229-1232. Haun, W., Coffman, A., Clasen, B.M., Demorest, Z.L., Lowy, A., Ray, E., Retterath, A., Stoddard, T., Juillerat, A., Cedrone, F., Mathis, L., Voytas, D.F., Zhang, F., (2014) Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family. Plant Biotechnol J 12, 934-940. Hsu, P.D., Lander, E.S., Zhang, F., (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262-1278. Jacobs, T.B., LaFayette, P.R., Schmitz, R.J., Parrott, W.A., (2015) Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnol 15, 16. Jiang, W., Zhou, H., Bi, H., Fromm, M., Yang, B., Weeks, D.P., (2013) Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 41, e188. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., Charpentier, E., (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821. Kay, S., Hahn, S., Marois, E., Hause, G., Bonas, U., (2007) A bacterial effector acts as a plant transcription factor and induces a cell size regulator. Science 318, 648-651. Kumar, V., Jain, M., (2015) The CRISPR-Cas system for plant genome editing: advances and opportunities. J Exp Bot 66, 47-57. Lei, Y., Lu, L., Liu, H.Y., Li, S., Xing, F., Chen, L.L., (2014) CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant 7, 1494-1496. Li, J.-F., Norville, J.E., Aach, J., McCormack, M., Zhang, D., Bush, J., Church, G.M., Sheen, J., (2013) Multiplex and homologous recombination-mediated genome editing 17

in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature biotechnology 31, 688-691. Li, T., Liu, B., Spalding, M.H., Weeks, D.P., Yang, B., (2012) High-efficiency TALEN-based gene editing produces disease-resistant rice. Nat Biotechnol 30, 390-392. Li, Z., Liu, Z.B., Xing, A., Moon, B.P., Koellhoffer, J.P., Huang, L., Ward, R.T., Clifton, E., Falco, S.C., Cigan, A.M., (2015) Cas9-guide RNA Directed Genome Editing in Soybean. Plant Physiol. Liang, C., Tian, J., Lam, H.M., Lim, B.L., Yan, X., Liao, H., (2010) Biochemical and molecular characterization of PvPAP3, a novel purple acid phosphatase isolated from common bean enhancing extracellular ATP utilization. Plant Physiol 152, 854-865. Mahfouz, M.M., Li, L., Shamimuzzaman, M., Wibowo, A., Fang, X., Zhu, J.K., (2011) De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks. Proc Natl Acad Sci U S A 108, 2623-2628. Mahfouz, M.M., Piatek, A., Stewart, C.N., Jr., (2014) Genome engineering via TALENs and CRISPR/Cas9 systems: challenges and perspectives. Plant Biotechnol J 12, 1006-1014. Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E., Church, G.M., (2013) RNA-guided human genome engineering via Cas9. Science 339, 823-826. Mao, Y., Zhang, H., Xu, N., Zhang, B., Gou, F., Zhu, J.K., (2013) Application of the CRISPR-Cas system for efficient genome engineering in plants. Mol Plant 6, 2008-2011. Maruyama, T., Dougan, S.K., Truttmann, M.C., Bilate, A.M., Ingram, J.R., Ploegh, H.L., (2015) Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol. Miao, J., Guo, D., Zhang, J., Huang, Q., Qin, G., Zhang, X., Wan, J., Gu, H., Qu, L.J., (2013) Targeted mutagenesis in rice using CRISPR-Cas system. Cell Res 23, 18

1233-1236. Moscou, M.J., Bogdanove, A.J., (2009) A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501-1501. Nekrasov, V., Staskawicz, B., Weigel, D., Jones, J.D., Kamoun, S., (2013) Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nature biotechnology 31, 691-693. Pattanayak, V., Lin, S., Guilinger, J.P., Ma, E., Doudna, J.A., Liu, D.R., (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature Biotechnology 31, 839-843. Ran, F.A., Cong, L., Yan, W.X., Scott, D.A., Gootenberg, J.S., Kriz, A.J., Zetsche, B., Shalem, O., Wu, X., Makarova, K.S., Koonin, E.V., Sharp, P.A., Zhang, F., (2015) In vivo genome editing using Staphylococcus aureus Cas9. Nature. Shalem, O., Sanjana, N.E., Zhang, F., (2015) High-throughput functional genomics using CRISPR-Cas9. Nat Rev Genet 16, 299-311. Shan, Q., Wang, Y., Chen, K., Liang, Z., Li, J., Zhang, Y., Zhang, K., Liu, J., Voytas, D.F., Zheng, X., Zhang, Y., Gao, C., (2013a) Rapid and efficient gene modification in rice and Brachypodium using TALENs. Mol Plant 6, 1365-1368. Shan, Q., Wang, Y., Li, J., Zhang, Y., Chen, K., Liang, Z., Zhang, K., Liu, J., Xi, J.J., Qiu, J.-L., (2013b) Targeted genome modification of crop plants using a CRISPR-Cas system. Nature biotechnology 31, 686-688. Sternberg, S.H., Doudna, J.A., (2015) Expanding the Biologist's Toolkit with CRISPR-Cas9. Mol Cell 58, 568-574. Sun, X., Hu, Z., Chen, R., Jiang, Q., Song, G., Zhang, H., Xi, Y., (2015) Targeted mutagenesis in soybean using the CRISPR-Cas9 system. Sci Rep 5, 10342. Tsai, S.Q., Wyvekens, N., Khayter, C., Foden, J.A., Thapar, V., Reyon, D., Goodwin, M.J., Aryee, M.J., Joung, J.K., (2014) Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol 32, 569-576. Voytas, D.F., (2013) Plant genome engineering with sequence-specific nucleases. 19

Annu Rev Plant Biol 64, 327-350. Voytas, D.F., Gao, C., (2014) Precision genome engineering and agriculture: opportunities and regulatory challenges. PLoS biology 12, e1001877. Wang, Y., Cheng, X., Shan, Q., Zhang, Y., Liu, J., Gao, C., Qiu, J.L., (2014) Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol 32, 947-951. Weinthal, D., Tovkach, A., Zeevi, V., Tzfira, T., (2010) Genome editing in plant cells by zinc finger nucleases. Trends Plant Sci 15, 308-321. Wiedenheft, B., Sternberg, S.H., Doudna, J.A., (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331-338. Wyman, C., Kanaar, R., (2006) DNA double-strand break repair: all's well that ends well. Annu Rev Genet 40, 363-383. Xie, K., Yang, Y., (2013) RNA-guided genome editing in plants using a CRISPR-Cas system. Mol Plant 6, 1975-1983. Zeng, P., Vadnais, D.A., Zhang, Z., Polacco, J.C., (2004) Refined glufosinate selection in Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill]. Plant Cell Rep 22, 478-482. Zhang, H., Zhang, J., Wei, P., Zhang, B., Gou, F., Feng, Z., Mao, Y., Yang, L., Zhang, H., Xu, N., Zhu, J.K., (2014) The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J 12, 797-807. Zhang, Y., Zhang, F., Li, X., Baller, J.A., Qi, Y., Starker, C.G., Bogdanove, A.J., Voytas, D.F., (2013) Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiol 161, 20-27.

20

Figure Captions

Figure 1 Four representative gels for analyzing the PCR products from hairy root containing with TALENs vectors or Cas9 vectors. Lane C1 and C2 are digested and undigested wild-type control, respectively. (A) TALENs-S1; (B) TALENs-S2; (C) Cas9-S12; (D) Cas9-S13. Nde I and Bcl I are typeⅡendonuclease, T7E1 and Cruiser are mismatch-sensitive endonuclease.

21

Figure 2 Sequences of some TALENs induced mutation representatives in hairy roots. Deletion and insertions are indicated by dashes and red letters, respectively.

22

Figure 3 Sequences of selected CRISPR/Cas9 induced mutations in hairy roots (PAM in green). Deletion and insertions are indicated by dashes and red letters, respectively.

23

Figure 4 Phenotypes of the pds mutants, induced by Cas9-D7 vector. (A) Non-transgenic wild-type adventitious bud; (B) and (C) mutant buds, red arrow: albino phenotype.

24

Tables

Table 1 Modification efficiency for hairy root events induced by TALENs. TALENs

Target Gene

ID

Total number

Number

of hairy roots

hairy

of roots

Frequency

Frequency (%) of

(%)

hairy roots with

with mutations D1

GmPDS11

80

GmPDS18

double mutations

11

17.5%

6.25% (5 double

16

20.0%

mutations)

S1

GmPDS11

67

13

19.4%

N.A.

S2

GmPDS11

71

15

21.1%

7.0% (5 double

S2-PDS18*

GmPDS18

71

5

7.0%

mutations)

N.A., not available. *The line “S2-PDS18” specifies off-target mutations at gene GmPDS18 caused by the S2 TALENs. As noted shown in the table, the S2 TALENs more frequently mutated GmPDS11, but also caused mutations at GmPDS18.

25

Table 2 Modification efficiency for hairy root events induced by Cas9/sgRNA system using Arabidopsis U6-26 promoter. Cas9 ID

Target Gene

Total number

Number

of

of hairy roots

hairy roots with

Frequency

Frequency (%) of

(%)

hairy toots with

mutations D7

GmPDS11

72

GmPDS18

double mutations

13

18.1%

12.5% (9 double

10

13.8%

mutation)

S11

GmPDS11

72

11

15.3%

N.A.

S12

GmPDS11

65

9

13.8%

N.A.

S13

GmPDS18

68

8

11.7%

N.A.

N.A., not available.

26

Table 3 Modification efficiency for hairy root events induced by Cas9/sgRNA system using soybean GmU6-16g-1 promoter. Cas9 ID

Target Gene

Total number

Number

of

of hairy roots

hairy roots with

Frequen

Frequency (%) of

cy (%)

hairy roots with

mutations D7-a*

GmPDS11

53

GmPDS18 D7-b*

GmPDS11 GmPDS18

54

double mutation

23

43.4%

43.4% (23 double

23

43.4%

mutation)

26

48.1%

48.1% (26 double

26

48.1%

mutation)

*The length of GmU6-16g-1 promoter is 616 bp (D7-a) and 350 bp (D7-b).

27