Accepted Manuscript Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System Zhen Liang, Kang Zhang, Kunling Chen, Caixia Gao PII:
S1673-8527(13)00197-5
DOI:
10.1016/j.jgg.2013.12.001
Reference:
JGG 248
To appear in:
Journal of Genetics and Genomics
Received Date: 12 November 2013 Revised Date:
5 December 2013
Accepted Date: 5 December 2013
Please cite this article as: Liang, Z., Zhang, K., Chen, K., Gao, C., Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System, Journal of Genetics and Genomics (2014), doi: 10.1016/ j.jgg.2013.12.001. 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.
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Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System
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Zhen Liang, Kang Zhang, Kunling Chen, Caixia Gao *
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State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of
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Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101,
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China
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Corresponding author. Tel & Fax: +86-10-6480 7727.
Email address：[email protected] (C. Gao).
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Abstract:
Transcription activator-like effector nucleases (TALENs) and clustered regularly
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interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas) systems
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have emerged as powerful tools for genome editing in a variety of species. Here, we
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report, for the first time, targeted mutagenesis in Zea mays using TALENs and the
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CRISPR/Cas system. We designed five TALENs targeting 4 genes, namely ZmPDS,
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ZmIPK1A, ZmIPK, ZmMRP4, and obtained targeting efficiencies of up to 23.1% in
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protoplasts, and about 13.3% to 39.1% of the transgenic plants were somatic
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mutations. Also, we constructed two gRNAs targeting the ZmIPK gene in maize
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protoplasts, at frequencies of 16.4% and 19.1%, respectively. In addition the
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CRISPR/Cas system induced targeted mutations in Zea mays protoplasts with
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efficiencies (13.1%) similar to those obtained with TALENs (9.1%). Our results show
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that both TALENs and the CRISPR/Cas system can be used for genome modification
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in maize.
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Key words: TAL-effector nucleases, CRISPR/Cas system, knock-out, Zea mays
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1. INTRODUCTION The development of whole genome sequencing and genomics has underlined the 1
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need for powerful genome editing tools to elucidate gene functions. In the past few
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years, artificial designed nucleases, including zinc-finger nucleases (ZFNs),
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transcription activator-like effector nucleases (TALENs) and clustered regularly
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interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas) systems
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have been widely used for targeted genome modification in plant species such as
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Arabidopsis (Zhang et al., 2010; Li et al., 2013; Qi et al., 2013), tobacco (Nekrasov et
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al., 2013; Zhang et al., 2013), rice (Li et al., 2012; Shan et al., 2013a, 2013b), barley
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(Wendt et al., 2013), soybean (Curtin et al., 2011), Brachypodium (Shan et al., 2013b)
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and maize (Shukla et al., 2009). All these nucleases consist of DNA binding domains
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together with non-specific nuclease domains that generate double-strand breaks
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(DSBs). The DSBs are mainly repaired by non-homologous end-joining (NHEJ) or
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homologous recombination (HR) pathway (Gaj et al., 2013). NHEJ simply rejoins the
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broken DNA ends in an error-prone fashion and often results in small deletions or
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insertions. In the HR pathway, DSBs are correctly repaired using a homologous donor
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DNA as template.
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TALENs and CRISPR/Cas systems have tremendous advantages over ZFNs,
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because of their one-to-one recognition of nucleotides, which makes them easier to
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design and construct (Boch et al., 2009; Cong et al., 2013; Mali et al., 2013). TALENs
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consist of customizable TALE DNA binding domains fused with non-specific Fok I
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cleavage domains. TALEs comprise a series of 34-amino-acid repeats, each with a
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repeat-variable di-residue (RVD) at positions 12 and 13 that can be used for
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recognizing a single target nucleotide (Fig. 1A). This modular architecture has been
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successfully produced by many different methods and makes TALENs an efficient
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procedure for genome editing (Cermak et al., 2011; Schmid-Burgk et al., 2012; Wang
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et al., 2012). The recently discovered type II CRISPR/Cas system requires a
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dual-tracrRNA:crRNA (gRNA) to guide the non-specific nuclease, Cas9, for DNA
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cleavage. The RuvC and HNH nuclease domains in Cas9 are responsible for cleaving
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the complementary DNA strands (Fig. 3A) (Gasiunas et al., 2012). The gRNA
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recognizes any genomic locus that is followed by a 5’-NGG protospacer-adjacent
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motif (PAM), and a 20-nt sequence preceding the PAM directs the Cas9 to cleave the
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target sequence by Watson-Crick base pairing. The simplicity of the cloning strategy
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and the fewer limitations of potential target sites make the CRISPR/Cas system very
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appealing. Maize (Zea mays) is an important model organism for fundamental research into
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the inheritance and functions of genes, and an important crop, yielding 12 billion
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bushels of grain in the USA alone in 2008. Phytic acid (PA), inositol 1, 2, 3, 4, 5,
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6-hexakisphosphate, is a natural product present in maize seeds, which represents
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about 75% of the total seed phosphorus. However, PA is an anti-nutritional compound,
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as it cannot be digested by monogastric animals and can cause environment pollution.
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Thus, it is important to reduce the PA content of maize seeds. We describe here the
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design and construction of TALENs and a gRNA:Cas9 construct that target the
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ZmIPK1A (Sun et al., 2007), ZmIPK (Shi et al., 2003), and ZmMRP4 (Shi et al., 2007)
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genes encoding enzymes that catalyze three steps in the phytic acid biosynthetic
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pathway. We demonstrate that both TALEN and CRISPR systems can introduce
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mutations at the target sites in transgenic maize. To our knowledge this is the first
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study to demonstrate targeted mutagenesis in maize using both TALENs and
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CRISPR/Cas systems. We anticipate that TALEN and CRISPR technologies will
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make targeted gene modification a routine practice in this economically important
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crop and substantially increase the potential for molecular breeding.
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2. RESULTS AND DISCUSSION Because plant tissue culture and transformation is time-consuming, it is necessary
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to test the activity of TALENs using a transient expression system before using them
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for genetic transformation. In this study, we carried out protoplast transformation
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based on previous work (Shan et al., 2013b) with slight modifications. Five pairs of
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TALENs (T-ZmPDS-1, T-ZmPDS-2, T-ZmIPK1A, T-ZmIPK and T-ZmMRP4) were
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generated targeting four endogenous loci, namely ZmPDS (NM_001111911),
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ZmIPK1A (EF447274), ZmIPK (AY172635), ZmMRP4 (EF586878), and each target
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site contained a unique restriction enzyme site within the spacer (Table S1). As the
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restriction enzyme sites were in the middle of the spacer, this approach roughly
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resembles mutagenesis caused by NHEJ repair. All of the TALEN repeat arrays were
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assembled by the Golden Gate assembly method and cloned into expression vector
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pZHY501 under the control of the 35S promoter (Zhang et al., 2013). Each pair of TALEN-encoding constructs was introduced into maize protoplasts by
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PEG-mediated transformation. After 40-48h cultivation, genomic DNA was isolated
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from the protoplasts and fragments encompassing the target sites were amplified by
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PCR. These were then digested with the corresponding restriction enzymes, visualized
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by agarose gel electrophoresis, and full length PCR products were then cloned and
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sequenced. A group of indels, with 2-17 bp deletions are shown in Fig. 1B.
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Efficiencies for T-ZmIPK and T-ZmMRP4 were 9.1% and 23.1%, respectively, as
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estimated from bands intensities. For T-ZmPDS-1, T-ZmPDS-2, and T-ZmIPK1A,
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enrichment PCR was carried out to detect mutations. The genomic DNAs were
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digested with the corresponding restriction enzymes (Table S1) and PCR was
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performed with primers flanking the target sites. After further digestion with the same
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restriction enzymes, undigested sequences in the samples incubated with TALENs
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carried mutations (Fig. S1).
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To evaluate whether TALENs can induce mutations in plants, all five pairs of TALENs
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Agrobacterium-mediated
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transformation. We carried out enrichment PCR to detect mutations induced by
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T-ZmIPK (Fig. 2A). We purified the undigested PCR products from individual plants
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and cloned them into pEasy-TI vector (Transgen, China). Ten clones were picked at
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random for sequence analysis. The results showed that different mutation patterns
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were present at the target sites (Fig. 2B). The mutations in other lines are shown in
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Fig. S2. With T-ZmIPK, about 39.1% of the independent plants generated mutations
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in ZmIPK, and similar results were obtained with T-ZmIPK1A, T-ZmPDS-1 and
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T-ZmPDS-2. The mutant sequences are presented in Fig. S2. Similar results have been
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obtained in barley (Wendt et al., 2013). The presence of somatic mutagenesis of T0
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plants may result from a delayed and sustained cleavage activity due to the TALENs.
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Different mutations may be transmitted to T1 plants by self-fertilization, so that T1
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plants derived from a single plant can carry different mutations. Studies in Xenopus
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embryos have shown that mosaic mutations in G0 are transmitted through the germ
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line (Lei et al., 2013). In summary, our data indicate that TALENs are able to bind to
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target sites and induce somatic mutations in Zea mays. Recent advances in understanding of the type II clustered regularly interspaced
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short palindromic repeats (CRISPR)/Cas system provide an easier approach to
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targeted mutations in eukaryotes (Cong et al., 2013; Mali et al., 2013). To optimize
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the CRISPR/Cas system in maize, we used the maize U3 promoter to drive gRNA
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expression. The codon-optimized Cas9 and gRNA scaffold have been described
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previously (Shan et al., 2013a).
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To see whether the CRISPR/Cas system induces mutations at target sites, we
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designed two gRNAs targeting the ZmIPK (AY172635) gene, preceded by an NGG,
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the protospacer adjacent motif (PAM). For ease of analysis, each gRNA had a
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restriction enzyme target site at the Cas9 cleavage site (Sac I for gRNA-1, BamH I for
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gRNA-2), 3bp before the PAM motif. We used the same strategy as above to detect
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mutations. Sequence analysis revealed 1 to 44 bp deletions and a 125 bp insertion (Fig.
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3B), confirming that the presence of mutations at the target sites. PCR/RE
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(PCR/Restriction enzyme assay) assays were performed to measure the mutation
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efficiency. The efficiencies at gRNA-1 and gRNA-2 were 16.4% and 19.1% (Fig. 3B),
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respectively. We also compared for the frequencies mutations induced by T-ZmIPK
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and gRNA-1, targeting the same site. As shown in Fig. 3C, CRISPR/Cas (13.1%) is
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capable of inducing mutations efficiently as TALENs (9.1%).
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In this study, we have demonstrated that TALENs and the CRISPR/Cas system are
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able to induce targeted mutagenesis in Zea mays with high and comparable
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efficiencies. Although TALENs are effective tools for genome editing, there are some
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limitations regarding the potential target sites, such as the need for T at position −1
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(Doyle et al., 2012) and the fact that some TALENs fail to cause mutations. The
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recently developed CRISPR/Cas system seems to provide a complementary approach
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to TALENs, as it only requires the PAM (NGG) motif preceding the recognition
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sequence. In addition, the CRISPR/Cas system has great advantages in terms of easy
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cloning and multiplex genome editing. However, the high-frequency of off-target
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mutations reported in human cells (not assessed in the present work) may seriously
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limit application of the CRISPR/Cas system (Fu et al., 2013; Pattanayak et al., 2013).
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Here, we conclude that both TALENs and CRISPR/Cas system can facilitate genome
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modification and functional genomic studies.
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3. MATERIALS AND METHODS
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3.1 Construction of TALENs
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The TALEN target sites used in this study were predicted by TAL
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Effector-Nucleotide Targeter 2.0 software (https://tale-nt.cac.cornell.edu/) (Doyle et
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al., 2012). The TALEN repeats were assembled using the Golden Gate strategy.
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TALEN Left/Right arrays were released by digestion with Xba I / BamH I, and
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successively cloned into the intermediate vector pZHY013 as Xba I / BamH I
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fragment and at Nhe I / Bgl II sites (Zhang et al., 2013). The entry vector was cloned
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into the destination vector, pGW6, which contains the bar gene as selectable marker
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and two 35S promoters to drive TALEN expression.
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3.2 Cas9 and gRNA design
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The Cas9 gene sequence used in this paper has been previously described (Shan et
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al., 2013a). The maize U3 promoter and the gRNA scaffold were amplified and
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cloned into pEasy Blunt vector (Transgen, China). The sequence of ZmU3-gRNA and
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the primers used are shown in Fig. S3 and Table S2. The synthesized target sequences
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were annealed and inserted into Bbs I-digested pU3-gRNA vector.
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3.3 Maize protoplast transformation
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Maize Hi-II seedlings were soaked in water overnight, and the seeds were
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transferred to Petri dishes between blotting papers for two days to germinate. The
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germinated seeds were then moved to the dark for 10−11 days at 24°C. Etiolated
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leaves were used in this study.
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To obtain mesophyll protoplasts, the middle parts of second leaves were cut into
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strips and digested with enzyme solution (1.5% Cellulase R10, 0.3% Macerozyme
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R10, 0.6 mol/L mannitol, 10 mmol/L MES at pH 5.7, 1 mmol/L CaCl2 and 0.1% 6
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BSA). A vacuum of −15~−20 (in Hg) was applied for 30 min, and the digestion was
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continued for 4−6 h with gentle shaking (90 rpm). The protoplasts were collected by
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filtration through a 40 µm nylon mesh and spun at 150 g for 3 min. After washing in
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W5 solution (154 mmol/L NaCl, 125 mmol/L CaCl2, 5 mmol/L KCl and 4 mmol/L
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MES at pH 5.7), the protoplasts were resuspended in MMg solution (0.4 mol/L
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mannitol, 15 mmol/L MgCl2 and 4 mmol/L MES at pH 5.7).
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The protoplast transformation procedure was modified from an Arabidopsis
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thaliana transformation protocol. Twenty µg of plasmid DNA (10 µg plasmid for each
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if for co-transformation) was incubated with 200 µL protoplasts in 220 µL of PEG
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solution at room temperature in the dark for 15 minutes. After adding 880 µL W5
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solution to stop the reaction, the protoplasts were harvested by centrifugation at 100 g
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for 3 minutes. They were resuspended in 2 mL WI solution (0.5 mol/L mannitol, 4
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mmol/L KCl and 4 mmol/L MES at pH 5.7) and incubated in 6-well plates in the dark
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at 22°C for 48 h.
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3.4 Detecting mutations by PCR/RE assays and Sanger sequencing
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Genomic DNA was extracted using the DNA Quick Plant System (Tiangen, China).
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To detect the efficiencies of T-ZmIPK, T-ZmMRP4, gRNA-1 and gRNA-2 in
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protoplasts, PCR/RE assay (PCR/Restriction enzyme assay) were carried out. We
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performed PCR with genomic DNA using primers flanking the target sites, then
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digested the PCR products with the appropriate restriction enzymes (Table S1) and
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visualized the products by agarose gel electrophoresis. The mutation frequencies were
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calculated by measuring band intensities with UVP Vision Works LS Image
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Acquisition Analysis Software 7.0. To detect the activities of T-ZmPDS-1,
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T-ZmPDS-2 and T-ZmIPK1A, we used enrichment PCR to reduce the amount of
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unaltered wild-type DNA in the samples. The genomic DNA was pre-digested at 37°C
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for 1 h (300−500 ng DNA, 1× Fastdigest buffer, 1 µL restriction enzyme), and PCR
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was performed on the predigested DNA. The amplified PCR products were digested
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with restriction enzymes at 37°C for 3 h, and after separation on 1.2% agarose gels,
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undigested bands were gel purified and cloned into pEasy-TI vector for Sanger
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sequencing.
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3.5 Agrobacterium transformation Maize Hi-II plants were grown in the field at the experimental station of Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,
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(Hainan province, China) under normal field conditions. Immature embryos isolated
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12−16 days after pollination were used for Agrobacterium-mediated transformation
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following the procedure of Ishida et al. (2007). Hygromycin-resistant plantlets
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regenerated after 9−12 weeks of selection, and about 30 to 50 transgenic lines were
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sampled from each transformation.
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ACKNOWLEGEMENTS
Our work was supported by the National Natural Science Foundation of China (Grant Nos. 383601 and 31200273).
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Figure Legends
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Fig. 1. Targeted mutagenesis using TALENs in maize protoplasts.
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A: Schematic representation of a TALEN binding to its target DNA (ZmIPK). The left
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and right binding sites are highlighted in red. The colored boxes represent the TALE
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effector repeats. Each recognizes one nucleotide and determines the sequence
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specificity. Fok I endonuclease (orange) is dimerized to cleave the target sequence. B:
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PCR/RE assays were performed to detect mutations at the ZmIPK and ZmMRP4 loci
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(left panel). Lane 1 and lane 2 represent PCR products of samples treated with the
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respective TALENs. Lanes 3 and lane 4 indicate digested and undigested wild-type
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controls, respectively. Representative sequences of the mutations induced by TALENs
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are shown in the right panel. The spacer and deletions are indicated by lower-case
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letters and black dots, respectively. The net change in length is shown to the right of
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each sequence (–, deletion).
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Fig. 2. TALEN-induced ZmIPK mutations in transgenic plants.
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A: A representative gel of the PCR products from independent transgenic seedlings. B:
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Sequence alignment of the WT (bottom) sequence and several independent amplicons
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from one plant. The TALE binding sites are indicated in red. The deletions are shown
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as black dots, and the net change in length is shown to the right of each sequence (–,
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deletion).
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Figure 3. Targeted mutagenesis via the CRISPR/Cas system in maize protoplasts.
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A: Schematic representation of the CRISPR/Cas system, including Cas9 and the
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gRNA. Cas9 flanked by two NLS signals is driven by two 35S promoters as described
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previously, and the gRNA is driven by the maize U3 promoter. PAM, the protospacer-
342
adjacent motif, is highlighted in red. B: PCR/RE assays were performed to detect
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mutations (left panel). Lane 1 and lane 2 represent PCR products of samples treated
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with the respective gRNA:Cas9. Lanes 3 and lane 4 represent digested and undigested
345
wild-type controls, respectively. Representative sequences of the mutations induced 11
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by the CRISPR/Cas system are shown in the right panel. The wild-type sequence is
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given at the bottom, with the target sites highlighted in red and the PAM highlighted
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in blue. The deletions are shown as black dots. The net change in length is shown to
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the right of each sequence (+, insertion; –, deletion). C: Comparison of the mutation
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efficiencies of TALEN and CRISPR/Cas system at the ZmIPK locus. Lane 1 and lane
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2 represent samples treated with Cas9 and gRNA-1. Lane 3 and 4 indicate samples
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treated with T-ZmIPK. Lane 5 and lane 6 represent the digested and undigested PCR
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product of the wild-type control, respectively. Indels (%) indicate mutagenesis
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frequencies of small insertions and deletions.
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Fig. S1. Targeted indel mutations induced by TALENs in maize protoplasts.
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Fig. S2. Sequences of TALEN-induced mutations in plants.
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Fig. S3. Full DNA sequence of the gRNA expression vector and pZmU3-gRNA.
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Table S1. Target lociand sequences of the mutations generated in maize by TALENs
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and the CRISPR/Cas system.
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Table S2. List of primers used in this study and their applications.
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Manuscript Title: Targeted Mutagenesis in Zea mays using TALENs and the CRISPR/Cas system
Figure S2. Sequences of TALEN-induced mutations in plants.
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Figure S1. Targeted indel mutations induced by TALENs in maize protoplasts.
Figure S3. Full DNA sequence of the gRNA expression vector and pZmU3-gRNA.
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Table S1. Target loci and sequences of the mutations generated in maize by TALENs and the CRISPR/Cas system.
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Table S2. List of primers used in this study, and their applications.
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Figure S1. Targeted indel mutation induced by TALENs in maize protoplasts. Lane1 and Lane2 represent the restriction enzyme digestion of the PCR products that treated with TALEN. Lane3 and Lane4 represent the digested and undigested PCR product of wild-type control, respectively. TALE binding sites and the spacer sequences are separately highlighted in red and lower case letters. Deletions are shown as black dots. The number of deletions for each sample is indicated on the right sides.
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Figure S2. Sequences of TALEN induced mutations in plants.
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>pZmU3-gRNA GAATTCCATCTAAGTATCTTGGTAAAGCATGGATTAATTTGGATGCCCACTTCAGGTCTATGCAGCTCCGGT GCCTTGTGATTGTGAGTTGTGACCGATGCTCATGCTATTCTGCATTTCTGCGATGTATGTAGCTAGTAGATC TTCAAAACTAACACCGCATGCCATCATCATCCACTGCTTGATTTTAGTCTCACCGCTGGCCAAAAATGTGAT
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GATGCCAGAAACCTCAACTACCTTGAATCAACACGGGCCCAACAGTGTGATGACGACAGAAACAAAAAAAA
ATGAGCCAATAGTTCAGAAGGAGGCACTATGCAGAAACTACATTTCTGAAGGTGACTAAAAGGTGAGCGTA GAGTGTAATTACTAGTAGTTTAGCCACCATTACCCAAATGCTTTCGAGCTTGTATTAAGATTTCCTAAGCTG
AGCATCATCACTGATCTGCAGGCCACCCTCGCTTCGCTGCCAAGATCAACAGCAACCATGTGGCGGCAACA
TCCAGCATTGCACATGGGCTAAAGATTGAGCTTTGTGCCTCGTCTAGGGATCAGCTGAGGTTATCAGTCTTT
CCTTTTTTTCATCCAGGTGAGGCATCAAGCTACTACTGCCTCGATTGGCTGGACCCGAAGCCCACATGTAGG
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ATACCAGAATGGGCCGACCCAGGACGCAGTATGTTGGCCAGTCCCACCGGTTAGTGCCATCTCGGTTGCTC ACATGCGTAGAAGCCAGCTTAAAAATTTAGCTTTGGTGACTCACAGCACTGTCTTCAACACAAGAAGACACG TTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCG
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GTGCTTTTTTTGGTACCGGATCCTACG
gRNA Target oligonucleotide 5’-CTTGNNNNNNNNNNNNNNNNNNN-3’ 3’-NNNNNNNNNNNNNNNNNNNCAAA-5’
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Figure S3. Full DNA sequence of the gRNA expression vector, pZmU3-gRNA. The ZmU3 promoter sequence and gRNA were highlighted in green and red respectively. The annealed gRNA target oligonucleotides were inserted into the two BbsI sites.
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Table S1. Target loci and sequences of the mutations generated in maize by TALEN and CRISPR-gRNA. TALEN ID
Accession No.
Target Site*
RE sites in spacer
ZmPDS
T-ZmPDS-1
NM_001111911
TGGTCAACCTTATGTTGAagctcaagatggcttaa cCGTTTCAGAATGGATGA
ZmPDS
T-ZmPDS-2
NM_001111911
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Gene Name
TGGATGGTAATCCGCCTGAaaggctatgcatgc
BpuEI: CTTAAG
SphI: GCTAGC
ctatTGTTGATCACATTCGGTCTA ZmIPK1A
T-ZmIPK1A
EF447274
TGCTGGGAGAGTAAATgcaagttcaattgataacA
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CTGCTGATGCCGCTCTTCTA ZmIPK
T-ZmIPK
AY172635
TCTCCATGCTCCAGGTcgtctccgagctcgaccaC
ZmMRP4
T-ZmMRP4
EF586878
TCCTTCGCGCTCTGCGttgtcattgcgtacgacgA
MfeI: CAATTG
SacI: GAGCTC
GCCGCCGACCAGGACA
BsiWI: CGTACG
gRNA-1
AY172635
ZmIPK
gRNA-2
AY172635
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CTCCAGGCGCCTGATA
CCGAGCTCGACCACGCCGCCGAC
SacI: GAGCTC
CCAGGGATCCGTCTCCTTCTCCC
BamHI: GGATCC
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Table S2. List of primers used in this study and their application. Primer sequence5’—3’
Usage
PDS 1F
GACAATAATAACATTACATCGTTAC
PCR/RE detection and sequencing
PDS 1R
TACAACTTTACATAGTACAAGCA
PDS 2F
TATATTAGTTGCCTGTTTTGTTTGA
PDS 2R
AGTTAAATTGGATAGCTGCCA
IPK1A 1F
AATTTATCTATAATCACCCCTAC
IPK1A 1R
TCTTGCTACAAGTGTTTCATC
IPK 1F
TCGCAGCCCCTGGCAGAGCAA
IPK 1R
GAGACCTGGGAGAAGGAGACGGATCC
IPK 2F
GCTTGTGCTCCAGGAGTTCGTC
IPK2R
CAGTCTCGTATCCTGGCATCTTGG
MRP4 1F
CCGTGCTGAGCTACGAGGTGG
MRP4 1R
CAGCAAATGTGCCATTGACCG
IPK gRNA 1F
AGCAGTCGGCGGCGTGGTCGAGCT
IPK gRNA 1R
AAACAGCTCGACCACGCCGCCGAC
IPK gRNA 2F
AGCAGGGAGAAGGAGACGGATCCC
IPK gRNA 2R
AAACGGGATCCGTCTCCTTCTCCC
ZmU3-F
GAATTCCATCTAAGTATCTTGGT
ZmU3-R
AGCCACGTGCTGTGAGTCACCAAAGCTAA
gRNA-F
TGTCTTCAACACAAGAAGACAC
gRNA-R
CGTAGGATCCGGTACCAAAAAAAGCACCGAC
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Primer name
PCR/RE detection and sequencing
PCR/RE detection and sequencing
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PCR/RE detection and sequencing
PCR/RE detection and sequencing
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PCR/RE detection and sequencing
gRNA Target oligonucleotide
gRNA Target oligonucleotide
pZmU3-gRNA construct
pZmU3-gRNA construct
Figure 1 Click here to download high resolution image
Figure 2 Click here to download high resolution image
Figure 3 Click here to download high resolution image
S1673-8527(13)00197-5
DOI:
10.1016/j.jgg.2013.12.001
Reference:
JGG 248
To appear in:
Journal of Genetics and Genomics
Received Date: 12 November 2013 Revised Date:
5 December 2013
Accepted Date: 5 December 2013
Please cite this article as: Liang, Z., Zhang, K., Chen, K., Gao, C., Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System, Journal of Genetics and Genomics (2014), doi: 10.1016/ j.jgg.2013.12.001. 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.
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Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System
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Zhen Liang, Kang Zhang, Kunling Chen, Caixia Gao *
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State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of
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Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101,
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China
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Corresponding author. Tel & Fax: +86-10-6480 7727.
Email address：[email protected] (C. Gao).
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Abstract:
Transcription activator-like effector nucleases (TALENs) and clustered regularly
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interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas) systems
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have emerged as powerful tools for genome editing in a variety of species. Here, we
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report, for the first time, targeted mutagenesis in Zea mays using TALENs and the
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CRISPR/Cas system. We designed five TALENs targeting 4 genes, namely ZmPDS,
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ZmIPK1A, ZmIPK, ZmMRP4, and obtained targeting efficiencies of up to 23.1% in
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protoplasts, and about 13.3% to 39.1% of the transgenic plants were somatic
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mutations. Also, we constructed two gRNAs targeting the ZmIPK gene in maize
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protoplasts, at frequencies of 16.4% and 19.1%, respectively. In addition the
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CRISPR/Cas system induced targeted mutations in Zea mays protoplasts with
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efficiencies (13.1%) similar to those obtained with TALENs (9.1%). Our results show
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that both TALENs and the CRISPR/Cas system can be used for genome modification
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in maize.
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Key words: TAL-effector nucleases, CRISPR/Cas system, knock-out, Zea mays
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1. INTRODUCTION The development of whole genome sequencing and genomics has underlined the 1
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need for powerful genome editing tools to elucidate gene functions. In the past few
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years, artificial designed nucleases, including zinc-finger nucleases (ZFNs),
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transcription activator-like effector nucleases (TALENs) and clustered regularly
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interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas) systems
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have been widely used for targeted genome modification in plant species such as
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Arabidopsis (Zhang et al., 2010; Li et al., 2013; Qi et al., 2013), tobacco (Nekrasov et
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al., 2013; Zhang et al., 2013), rice (Li et al., 2012; Shan et al., 2013a, 2013b), barley
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(Wendt et al., 2013), soybean (Curtin et al., 2011), Brachypodium (Shan et al., 2013b)
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and maize (Shukla et al., 2009). All these nucleases consist of DNA binding domains
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together with non-specific nuclease domains that generate double-strand breaks
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(DSBs). The DSBs are mainly repaired by non-homologous end-joining (NHEJ) or
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homologous recombination (HR) pathway (Gaj et al., 2013). NHEJ simply rejoins the
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broken DNA ends in an error-prone fashion and often results in small deletions or
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insertions. In the HR pathway, DSBs are correctly repaired using a homologous donor
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DNA as template.
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TALENs and CRISPR/Cas systems have tremendous advantages over ZFNs,
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because of their one-to-one recognition of nucleotides, which makes them easier to
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design and construct (Boch et al., 2009; Cong et al., 2013; Mali et al., 2013). TALENs
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consist of customizable TALE DNA binding domains fused with non-specific Fok I
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cleavage domains. TALEs comprise a series of 34-amino-acid repeats, each with a
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repeat-variable di-residue (RVD) at positions 12 and 13 that can be used for
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recognizing a single target nucleotide (Fig. 1A). This modular architecture has been
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successfully produced by many different methods and makes TALENs an efficient
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procedure for genome editing (Cermak et al., 2011; Schmid-Burgk et al., 2012; Wang
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et al., 2012). The recently discovered type II CRISPR/Cas system requires a
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dual-tracrRNA:crRNA (gRNA) to guide the non-specific nuclease, Cas9, for DNA
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cleavage. The RuvC and HNH nuclease domains in Cas9 are responsible for cleaving
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the complementary DNA strands (Fig. 3A) (Gasiunas et al., 2012). The gRNA
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recognizes any genomic locus that is followed by a 5’-NGG protospacer-adjacent
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motif (PAM), and a 20-nt sequence preceding the PAM directs the Cas9 to cleave the
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target sequence by Watson-Crick base pairing. The simplicity of the cloning strategy
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and the fewer limitations of potential target sites make the CRISPR/Cas system very
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appealing. Maize (Zea mays) is an important model organism for fundamental research into
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the inheritance and functions of genes, and an important crop, yielding 12 billion
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bushels of grain in the USA alone in 2008. Phytic acid (PA), inositol 1, 2, 3, 4, 5,
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6-hexakisphosphate, is a natural product present in maize seeds, which represents
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about 75% of the total seed phosphorus. However, PA is an anti-nutritional compound,
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as it cannot be digested by monogastric animals and can cause environment pollution.
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Thus, it is important to reduce the PA content of maize seeds. We describe here the
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design and construction of TALENs and a gRNA:Cas9 construct that target the
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ZmIPK1A (Sun et al., 2007), ZmIPK (Shi et al., 2003), and ZmMRP4 (Shi et al., 2007)
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genes encoding enzymes that catalyze three steps in the phytic acid biosynthetic
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pathway. We demonstrate that both TALEN and CRISPR systems can introduce
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mutations at the target sites in transgenic maize. To our knowledge this is the first
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study to demonstrate targeted mutagenesis in maize using both TALENs and
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CRISPR/Cas systems. We anticipate that TALEN and CRISPR technologies will
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make targeted gene modification a routine practice in this economically important
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crop and substantially increase the potential for molecular breeding.
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2. RESULTS AND DISCUSSION Because plant tissue culture and transformation is time-consuming, it is necessary
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to test the activity of TALENs using a transient expression system before using them
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for genetic transformation. In this study, we carried out protoplast transformation
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based on previous work (Shan et al., 2013b) with slight modifications. Five pairs of
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TALENs (T-ZmPDS-1, T-ZmPDS-2, T-ZmIPK1A, T-ZmIPK and T-ZmMRP4) were
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generated targeting four endogenous loci, namely ZmPDS (NM_001111911),
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ZmIPK1A (EF447274), ZmIPK (AY172635), ZmMRP4 (EF586878), and each target
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site contained a unique restriction enzyme site within the spacer (Table S1). As the
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restriction enzyme sites were in the middle of the spacer, this approach roughly
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resembles mutagenesis caused by NHEJ repair. All of the TALEN repeat arrays were
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assembled by the Golden Gate assembly method and cloned into expression vector
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pZHY501 under the control of the 35S promoter (Zhang et al., 2013). Each pair of TALEN-encoding constructs was introduced into maize protoplasts by
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PEG-mediated transformation. After 40-48h cultivation, genomic DNA was isolated
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from the protoplasts and fragments encompassing the target sites were amplified by
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PCR. These were then digested with the corresponding restriction enzymes, visualized
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by agarose gel electrophoresis, and full length PCR products were then cloned and
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sequenced. A group of indels, with 2-17 bp deletions are shown in Fig. 1B.
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Efficiencies for T-ZmIPK and T-ZmMRP4 were 9.1% and 23.1%, respectively, as
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estimated from bands intensities. For T-ZmPDS-1, T-ZmPDS-2, and T-ZmIPK1A,
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enrichment PCR was carried out to detect mutations. The genomic DNAs were
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digested with the corresponding restriction enzymes (Table S1) and PCR was
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performed with primers flanking the target sites. After further digestion with the same
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restriction enzymes, undigested sequences in the samples incubated with TALENs
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carried mutations (Fig. S1).
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To evaluate whether TALENs can induce mutations in plants, all five pairs of TALENs
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introduced
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Hi-II
plants
by
Agrobacterium-mediated
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transformation. We carried out enrichment PCR to detect mutations induced by
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T-ZmIPK (Fig. 2A). We purified the undigested PCR products from individual plants
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and cloned them into pEasy-TI vector (Transgen, China). Ten clones were picked at
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random for sequence analysis. The results showed that different mutation patterns
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were present at the target sites (Fig. 2B). The mutations in other lines are shown in
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Fig. S2. With T-ZmIPK, about 39.1% of the independent plants generated mutations
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in ZmIPK, and similar results were obtained with T-ZmIPK1A, T-ZmPDS-1 and
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T-ZmPDS-2. The mutant sequences are presented in Fig. S2. Similar results have been
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obtained in barley (Wendt et al., 2013). The presence of somatic mutagenesis of T0
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plants may result from a delayed and sustained cleavage activity due to the TALENs.
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Different mutations may be transmitted to T1 plants by self-fertilization, so that T1
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plants derived from a single plant can carry different mutations. Studies in Xenopus
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embryos have shown that mosaic mutations in G0 are transmitted through the germ
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line (Lei et al., 2013). In summary, our data indicate that TALENs are able to bind to
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target sites and induce somatic mutations in Zea mays. Recent advances in understanding of the type II clustered regularly interspaced
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short palindromic repeats (CRISPR)/Cas system provide an easier approach to
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targeted mutations in eukaryotes (Cong et al., 2013; Mali et al., 2013). To optimize
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the CRISPR/Cas system in maize, we used the maize U3 promoter to drive gRNA
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expression. The codon-optimized Cas9 and gRNA scaffold have been described
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previously (Shan et al., 2013a).
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To see whether the CRISPR/Cas system induces mutations at target sites, we
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designed two gRNAs targeting the ZmIPK (AY172635) gene, preceded by an NGG,
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the protospacer adjacent motif (PAM). For ease of analysis, each gRNA had a
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restriction enzyme target site at the Cas9 cleavage site (Sac I for gRNA-1, BamH I for
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gRNA-2), 3bp before the PAM motif. We used the same strategy as above to detect
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mutations. Sequence analysis revealed 1 to 44 bp deletions and a 125 bp insertion (Fig.
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3B), confirming that the presence of mutations at the target sites. PCR/RE
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(PCR/Restriction enzyme assay) assays were performed to measure the mutation
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efficiency. The efficiencies at gRNA-1 and gRNA-2 were 16.4% and 19.1% (Fig. 3B),
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respectively. We also compared for the frequencies mutations induced by T-ZmIPK
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and gRNA-1, targeting the same site. As shown in Fig. 3C, CRISPR/Cas (13.1%) is
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capable of inducing mutations efficiently as TALENs (9.1%).
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In this study, we have demonstrated that TALENs and the CRISPR/Cas system are
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able to induce targeted mutagenesis in Zea mays with high and comparable
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efficiencies. Although TALENs are effective tools for genome editing, there are some
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limitations regarding the potential target sites, such as the need for T at position −1
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(Doyle et al., 2012) and the fact that some TALENs fail to cause mutations. The
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recently developed CRISPR/Cas system seems to provide a complementary approach
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to TALENs, as it only requires the PAM (NGG) motif preceding the recognition
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sequence. In addition, the CRISPR/Cas system has great advantages in terms of easy
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cloning and multiplex genome editing. However, the high-frequency of off-target
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mutations reported in human cells (not assessed in the present work) may seriously
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limit application of the CRISPR/Cas system (Fu et al., 2013; Pattanayak et al., 2013).
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Here, we conclude that both TALENs and CRISPR/Cas system can facilitate genome
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modification and functional genomic studies.
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3. MATERIALS AND METHODS
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3.1 Construction of TALENs
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The TALEN target sites used in this study were predicted by TAL
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Effector-Nucleotide Targeter 2.0 software (https://tale-nt.cac.cornell.edu/) (Doyle et
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al., 2012). The TALEN repeats were assembled using the Golden Gate strategy.
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TALEN Left/Right arrays were released by digestion with Xba I / BamH I, and
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successively cloned into the intermediate vector pZHY013 as Xba I / BamH I
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fragment and at Nhe I / Bgl II sites (Zhang et al., 2013). The entry vector was cloned
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into the destination vector, pGW6, which contains the bar gene as selectable marker
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and two 35S promoters to drive TALEN expression.
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3.2 Cas9 and gRNA design
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The Cas9 gene sequence used in this paper has been previously described (Shan et
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al., 2013a). The maize U3 promoter and the gRNA scaffold were amplified and
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cloned into pEasy Blunt vector (Transgen, China). The sequence of ZmU3-gRNA and
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the primers used are shown in Fig. S3 and Table S2. The synthesized target sequences
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were annealed and inserted into Bbs I-digested pU3-gRNA vector.
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3.3 Maize protoplast transformation
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Maize Hi-II seedlings were soaked in water overnight, and the seeds were
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transferred to Petri dishes between blotting papers for two days to germinate. The
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germinated seeds were then moved to the dark for 10−11 days at 24°C. Etiolated
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leaves were used in this study.
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To obtain mesophyll protoplasts, the middle parts of second leaves were cut into
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strips and digested with enzyme solution (1.5% Cellulase R10, 0.3% Macerozyme
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R10, 0.6 mol/L mannitol, 10 mmol/L MES at pH 5.7, 1 mmol/L CaCl2 and 0.1% 6
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BSA). A vacuum of −15~−20 (in Hg) was applied for 30 min, and the digestion was
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continued for 4−6 h with gentle shaking (90 rpm). The protoplasts were collected by
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filtration through a 40 µm nylon mesh and spun at 150 g for 3 min. After washing in
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W5 solution (154 mmol/L NaCl, 125 mmol/L CaCl2, 5 mmol/L KCl and 4 mmol/L
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MES at pH 5.7), the protoplasts were resuspended in MMg solution (0.4 mol/L
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mannitol, 15 mmol/L MgCl2 and 4 mmol/L MES at pH 5.7).
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The protoplast transformation procedure was modified from an Arabidopsis
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thaliana transformation protocol. Twenty µg of plasmid DNA (10 µg plasmid for each
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if for co-transformation) was incubated with 200 µL protoplasts in 220 µL of PEG
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solution at room temperature in the dark for 15 minutes. After adding 880 µL W5
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solution to stop the reaction, the protoplasts were harvested by centrifugation at 100 g
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for 3 minutes. They were resuspended in 2 mL WI solution (0.5 mol/L mannitol, 4
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mmol/L KCl and 4 mmol/L MES at pH 5.7) and incubated in 6-well plates in the dark
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at 22°C for 48 h.
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3.4 Detecting mutations by PCR/RE assays and Sanger sequencing
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Genomic DNA was extracted using the DNA Quick Plant System (Tiangen, China).
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To detect the efficiencies of T-ZmIPK, T-ZmMRP4, gRNA-1 and gRNA-2 in
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protoplasts, PCR/RE assay (PCR/Restriction enzyme assay) were carried out. We
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performed PCR with genomic DNA using primers flanking the target sites, then
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digested the PCR products with the appropriate restriction enzymes (Table S1) and
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visualized the products by agarose gel electrophoresis. The mutation frequencies were
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calculated by measuring band intensities with UVP Vision Works LS Image
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Acquisition Analysis Software 7.0. To detect the activities of T-ZmPDS-1,
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T-ZmPDS-2 and T-ZmIPK1A, we used enrichment PCR to reduce the amount of
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unaltered wild-type DNA in the samples. The genomic DNA was pre-digested at 37°C
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for 1 h (300−500 ng DNA, 1× Fastdigest buffer, 1 µL restriction enzyme), and PCR
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was performed on the predigested DNA. The amplified PCR products were digested
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with restriction enzymes at 37°C for 3 h, and after separation on 1.2% agarose gels,
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undigested bands were gel purified and cloned into pEasy-TI vector for Sanger
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sequencing.
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3.5 Agrobacterium transformation Maize Hi-II plants were grown in the field at the experimental station of Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,
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(Hainan province, China) under normal field conditions. Immature embryos isolated
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12−16 days after pollination were used for Agrobacterium-mediated transformation
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following the procedure of Ishida et al. (2007). Hygromycin-resistant plantlets
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regenerated after 9−12 weeks of selection, and about 30 to 50 transgenic lines were
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sampled from each transformation.
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ACKNOWLEGEMENTS
Our work was supported by the National Natural Science Foundation of China (Grant Nos. 383601 and 31200273).
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in Sanya
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Mitchell, J.C., Arnold, N.L., Gopalan, S., Meng, X., Choi, V.M., Rock, J.M., Wu, Y.Y., Katibah, G.E., Zhifang, G., McCaskill, D., Simpson, M.A., Blakeslee, B., Greenwalt, S.A., Butler, H.J., Hinkley, S.J., Zhang, L., Rebar, E.J., Gregory, P.D., Urnov, F.D., 2009. Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459, 437-441. Sun, Y., Thompson, M., Lin, G., Butler, H., Gao, Z., Thornburgh, S., Yau, K., Smith, D.A., Shukla, V.K., 2007. Inositol 1,3,4,5,6-pentakisphosphate 2-kinase from maize: molecular and biochemical characterization. Plant Physiol. 144, 1278-1291. Wang, Z., Li, J., Huang, H., Wang, G. C., Jiang, M. J., Yin, S. Y., Sun, C. H., Zhang, H. S., Zhuang, F. F., Xi, J. J., 2012. An integrated chip for the high-throughput synthesis of transcription activator-like effectors. Angew. Chem. Int. Ed. 51, 8505 –8508. Wendt, T., Holm, P.B., Starker, C.G., Christian, M., Voytas, D.F., Brinch-Pedersen, H., Holme, I.B., 2013. TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants. Plant Mol. Biol. 83, 279-285. Zhang, F., Maeder, M.L., Unger-Wallace, E., Hoshaw, J.P., Reyon, D., Christian, M., Li, X., Pierick, C.J., Dobbs, D., Peterson, T., Joung, J.K., Voytas, D.F., 2010. High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases. Proc. Natl. Acad. Sci. USA 107, 12028-12033. 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.
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Figure Legends
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Fig. 1. Targeted mutagenesis using TALENs in maize protoplasts.
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A: Schematic representation of a TALEN binding to its target DNA (ZmIPK). The left
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and right binding sites are highlighted in red. The colored boxes represent the TALE
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effector repeats. Each recognizes one nucleotide and determines the sequence
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specificity. Fok I endonuclease (orange) is dimerized to cleave the target sequence. B:
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PCR/RE assays were performed to detect mutations at the ZmIPK and ZmMRP4 loci
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(left panel). Lane 1 and lane 2 represent PCR products of samples treated with the
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respective TALENs. Lanes 3 and lane 4 indicate digested and undigested wild-type
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controls, respectively. Representative sequences of the mutations induced by TALENs
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are shown in the right panel. The spacer and deletions are indicated by lower-case
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letters and black dots, respectively. The net change in length is shown to the right of
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each sequence (–, deletion).
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Fig. 2. TALEN-induced ZmIPK mutations in transgenic plants.
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A: A representative gel of the PCR products from independent transgenic seedlings. B:
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Sequence alignment of the WT (bottom) sequence and several independent amplicons
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from one plant. The TALE binding sites are indicated in red. The deletions are shown
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as black dots, and the net change in length is shown to the right of each sequence (–,
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deletion).
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Figure 3. Targeted mutagenesis via the CRISPR/Cas system in maize protoplasts.
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A: Schematic representation of the CRISPR/Cas system, including Cas9 and the
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gRNA. Cas9 flanked by two NLS signals is driven by two 35S promoters as described
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previously, and the gRNA is driven by the maize U3 promoter. PAM, the protospacer-
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adjacent motif, is highlighted in red. B: PCR/RE assays were performed to detect
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mutations (left panel). Lane 1 and lane 2 represent PCR products of samples treated
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with the respective gRNA:Cas9. Lanes 3 and lane 4 represent digested and undigested
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wild-type controls, respectively. Representative sequences of the mutations induced 11
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by the CRISPR/Cas system are shown in the right panel. The wild-type sequence is
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given at the bottom, with the target sites highlighted in red and the PAM highlighted
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in blue. The deletions are shown as black dots. The net change in length is shown to
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the right of each sequence (+, insertion; –, deletion). C: Comparison of the mutation
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efficiencies of TALEN and CRISPR/Cas system at the ZmIPK locus. Lane 1 and lane
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2 represent samples treated with Cas9 and gRNA-1. Lane 3 and 4 indicate samples
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treated with T-ZmIPK. Lane 5 and lane 6 represent the digested and undigested PCR
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product of the wild-type control, respectively. Indels (%) indicate mutagenesis
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frequencies of small insertions and deletions.
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Fig. S1. Targeted indel mutations induced by TALENs in maize protoplasts.
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Fig. S2. Sequences of TALEN-induced mutations in plants.
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Fig. S3. Full DNA sequence of the gRNA expression vector and pZmU3-gRNA.
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Table S1. Target lociand sequences of the mutations generated in maize by TALENs
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and the CRISPR/Cas system.
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Table S2. List of primers used in this study and their applications.
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Manuscript Title: Targeted Mutagenesis in Zea mays using TALENs and the CRISPR/Cas system
Figure S2. Sequences of TALEN-induced mutations in plants.
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Figure S1. Targeted indel mutations induced by TALENs in maize protoplasts.
Figure S3. Full DNA sequence of the gRNA expression vector and pZmU3-gRNA.
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Table S1. Target loci and sequences of the mutations generated in maize by TALENs and the CRISPR/Cas system.
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Table S2. List of primers used in this study, and their applications.
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Figure S1. Targeted indel mutation induced by TALENs in maize protoplasts. Lane1 and Lane2 represent the restriction enzyme digestion of the PCR products that treated with TALEN. Lane3 and Lane4 represent the digested and undigested PCR product of wild-type control, respectively. TALE binding sites and the spacer sequences are separately highlighted in red and lower case letters. Deletions are shown as black dots. The number of deletions for each sample is indicated on the right sides.
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Figure S2. Sequences of TALEN induced mutations in plants.
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>pZmU3-gRNA GAATTCCATCTAAGTATCTTGGTAAAGCATGGATTAATTTGGATGCCCACTTCAGGTCTATGCAGCTCCGGT GCCTTGTGATTGTGAGTTGTGACCGATGCTCATGCTATTCTGCATTTCTGCGATGTATGTAGCTAGTAGATC TTCAAAACTAACACCGCATGCCATCATCATCCACTGCTTGATTTTAGTCTCACCGCTGGCCAAAAATGTGAT
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GATGCCAGAAACCTCAACTACCTTGAATCAACACGGGCCCAACAGTGTGATGACGACAGAAACAAAAAAAA
ATGAGCCAATAGTTCAGAAGGAGGCACTATGCAGAAACTACATTTCTGAAGGTGACTAAAAGGTGAGCGTA GAGTGTAATTACTAGTAGTTTAGCCACCATTACCCAAATGCTTTCGAGCTTGTATTAAGATTTCCTAAGCTG
AGCATCATCACTGATCTGCAGGCCACCCTCGCTTCGCTGCCAAGATCAACAGCAACCATGTGGCGGCAACA
TCCAGCATTGCACATGGGCTAAAGATTGAGCTTTGTGCCTCGTCTAGGGATCAGCTGAGGTTATCAGTCTTT
CCTTTTTTTCATCCAGGTGAGGCATCAAGCTACTACTGCCTCGATTGGCTGGACCCGAAGCCCACATGTAGG
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ATACCAGAATGGGCCGACCCAGGACGCAGTATGTTGGCCAGTCCCACCGGTTAGTGCCATCTCGGTTGCTC ACATGCGTAGAAGCCAGCTTAAAAATTTAGCTTTGGTGACTCACAGCACTGTCTTCAACACAAGAAGACACG TTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCG
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GTGCTTTTTTTGGTACCGGATCCTACG
gRNA Target oligonucleotide 5’-CTTGNNNNNNNNNNNNNNNNNNN-3’ 3’-NNNNNNNNNNNNNNNNNNNCAAA-5’
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Figure S3. Full DNA sequence of the gRNA expression vector, pZmU3-gRNA. The ZmU3 promoter sequence and gRNA were highlighted in green and red respectively. The annealed gRNA target oligonucleotides were inserted into the two BbsI sites.
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Table S1. Target loci and sequences of the mutations generated in maize by TALEN and CRISPR-gRNA. TALEN ID
Accession No.
Target Site*
RE sites in spacer
ZmPDS
T-ZmPDS-1
NM_001111911
TGGTCAACCTTATGTTGAagctcaagatggcttaa cCGTTTCAGAATGGATGA
ZmPDS
T-ZmPDS-2
NM_001111911
RI PT
Gene Name
TGGATGGTAATCCGCCTGAaaggctatgcatgc
BpuEI: CTTAAG
SphI: GCTAGC
ctatTGTTGATCACATTCGGTCTA ZmIPK1A
T-ZmIPK1A
EF447274
TGCTGGGAGAGTAAATgcaagttcaattgataacA
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CTGCTGATGCCGCTCTTCTA ZmIPK
T-ZmIPK
AY172635
TCTCCATGCTCCAGGTcgtctccgagctcgaccaC
ZmMRP4
T-ZmMRP4
EF586878
TCCTTCGCGCTCTGCGttgtcattgcgtacgacgA
MfeI: CAATTG
SacI: GAGCTC
GCCGCCGACCAGGACA
BsiWI: CGTACG
gRNA-1
AY172635
ZmIPK
gRNA-2
AY172635
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CTCCAGGCGCCTGATA
CCGAGCTCGACCACGCCGCCGAC
SacI: GAGCTC
CCAGGGATCCGTCTCCTTCTCCC
BamHI: GGATCC
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Table S2. List of primers used in this study and their application. Primer sequence5’—3’
Usage
PDS 1F
GACAATAATAACATTACATCGTTAC
PCR/RE detection and sequencing
PDS 1R
TACAACTTTACATAGTACAAGCA
PDS 2F
TATATTAGTTGCCTGTTTTGTTTGA
PDS 2R
AGTTAAATTGGATAGCTGCCA
IPK1A 1F
AATTTATCTATAATCACCCCTAC
IPK1A 1R
TCTTGCTACAAGTGTTTCATC
IPK 1F
TCGCAGCCCCTGGCAGAGCAA
IPK 1R
GAGACCTGGGAGAAGGAGACGGATCC
IPK 2F
GCTTGTGCTCCAGGAGTTCGTC
IPK2R
CAGTCTCGTATCCTGGCATCTTGG
MRP4 1F
CCGTGCTGAGCTACGAGGTGG
MRP4 1R
CAGCAAATGTGCCATTGACCG
IPK gRNA 1F
AGCAGTCGGCGGCGTGGTCGAGCT
IPK gRNA 1R
AAACAGCTCGACCACGCCGCCGAC
IPK gRNA 2F
AGCAGGGAGAAGGAGACGGATCCC
IPK gRNA 2R
AAACGGGATCCGTCTCCTTCTCCC
ZmU3-F
GAATTCCATCTAAGTATCTTGGT
ZmU3-R
AGCCACGTGCTGTGAGTCACCAAAGCTAA
gRNA-F
TGTCTTCAACACAAGAAGACAC
gRNA-R
CGTAGGATCCGGTACCAAAAAAAGCACCGAC
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Primer name
PCR/RE detection and sequencing
PCR/RE detection and sequencing
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PCR/RE detection and sequencing
PCR/RE detection and sequencing
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PCR/RE detection and sequencing
gRNA Target oligonucleotide
gRNA Target oligonucleotide
pZmU3-gRNA construct
pZmU3-gRNA construct
Figure 1 Click here to download high resolution image
Figure 2 Click here to download high resolution image
Figure 3 Click here to download high resolution image
