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Cas9 System
Accepted Manuscript Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System Chao Feng, Jing Yuan, Rui Wang, Yang Liu, James A. Birchler, Fangpu Han PII:
S1673-8527(15)00179-4
DOI:
10.1016/j.jgg.2015.10.002
Reference:
JGG 408
To appear in:
Journal of Genetics and Genomics
Received Date: 16 August 2015 Revised Date:
14 October 2015
Accepted Date: 20 October 2015
Please cite this article as: Feng, C., Yuan, J., Wang, R., Liu, Y., Birchler, J.A., Han, F., Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System, Journal of Genetics and Genomics (2015), doi: 10.1016/j.jgg.2015.10.002. 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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Efficient Targeted Genome Modification in Maize Using
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CRISPR/Cas9 System
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Chao Fenga,b,1, Jing Yuana,1, Rui Wanga , Yang Liua,b, James A. Birchlerc and Fangpu
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Hana,*
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Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
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University of Chinese Academy of Sciences, Beijing 100049, China
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Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400, USA
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These authors contributed equally to this work.
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Tel: +86 10 6480 7926, fax: +86 10 6485 4467
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E-mail address: [email protected]
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Correspondending author:
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State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and
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ABSTRACT
31 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system, which is a
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newly developed technology for targeted genome modification, has been successfully used in a
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number of species. In this study, we applied this technology to carry out targeted genome
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modification in maize. A marker gene Zmzb7 was chosen for targeting. The sgRNA-Cas9
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construct was transformed into maize protoplasts, and indel mutations could be detected. A mutant
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seedling with an expected albino phenotype was obtained from screening 120 seedlings generated
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from 10 callus events. Mutation efficiency in maize heterochromatic regions was also investigated.
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Twelve sites with different expression levels in maize centromeres or pericentromere regions were
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selected. The sgRNA-Cas9 constructs were transformed into protoplasts followed by sequencing
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the transformed protoplast genomic DNA. The results show that the genes in heterochromatic
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regions could be targeted by the CRISPR/Cas9 system efficiently, no matter whether they are
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expressed or not. Meanwhile, off-target mutations were not found in the similar sites having no
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PAM motif or having more than two mismatches. Together, our results show that the
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CRISPR/Cas9 system is a robust and efficient tool for genome modification in both euchromatic
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and heterochromatic regions in maize.
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Keywords: CRISPR/Cas9; targeted genome modification; heterochromatic region; maize
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INTRODUCTION
63 As one of the most important crops in the world, maize (Zea mays) is also a model plant for
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genetic research. Traditional strategies for genetic study of maize mostly depend on chemical
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(EMS) treatment or transposon tagging to generate mutant alleles (Candela and Hake, 2008).
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These methods have been proved to be effective, but because the mutations occur randomly, it
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takes time and energy to perform a large screen.
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“Targeted genome modification” is a new concept. The basic principle is to design a nuclease
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that can specifically target a genomic locus, generate a DSB (double strand break) at the site
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(Puchta and Fauser, 2013), and then use the endogenous cellular DSB repair system to modify the
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targeted genomic locus. Two artificial nucleases, ZFN (zinc finger nuclease) and TALEN
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(transcription activator like effector nuclease) were successfully used for targeted genome
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modifications in the past, especially TALENs (Carroll, 2011; Joung and Sander, 2012;
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Perez-Pinera et al., 2012). More recently, a new “targeted genome modification” system, CRISPR
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(clustered regularly interspaced short palindromic repeats)/Cas9 system, was developed and
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quickly spread in the scientific community due to its simple cloning procedure and relatively
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higher efficiency (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013). For the CRISPR/Cas9
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system, the endonuclease Cas9 is guided by a single guide RNA (sgRNA), which includes two
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parts. One of them (usually 20 bp) recognizes the target sites through Watson-Crick base pairing
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between DNA and RNA, and the other forms a structure that recruits the Cas9 endonuclease. Only
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a PAM (NGG) sequence is necessary for target site design, which means almost all genes in a
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genome could be targeted by this system (Hsu et al., 2014). The utilization of CRISPR/Cas9
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system for genome editing has been reported in various species, from bacteria to mammals, which
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supports the point that it hardly has species limits (Bassett an Liu, 2014; Cho et al., 2013; Hwang
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et al., 2013; Jiang et al., 2013; Li et al., 2013a; Li et al., 2013b; Nekrasov et al., 2013; Shan et al.,
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2013). In maize, genome modification by the CRISPR/Cas9 system has been reported previously
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(Liang et al., 2014; Xing Hui-Li, 2014). Mutations mediated by the CRISPR/Cas9 system have
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been detected in maize protoplasts and transgenic seedlings.
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ACCEPTED MANUSCRIPT Although the CRISPR/Cas9 system shows high efficiency for targeted genome modification, it
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did not work well constitutively. In some cases, the target sites meeting the requirements for
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targeting cannot be targeted (Hwang et al., 2013). The reason is unknown, and the molecular
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mechanisms for Cas9 nuclease to cut the target DNA sequence are to be explored. As the process
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of gene targeting by CRIPSR/Cas9 system depends on the sgRNA to search and target to the
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specific site, it is an interesting question of whether the chromatin environment would influence
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the sgRNA binding ability and change the gene targeting efficiency in plants.
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Here, we report our results of using CRIPSR/Cas9 system to do targeted genome modification
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in maize. First, a marker gene Zmzb7 was successfully mutated, and a mutant plant exhibiting the
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expected phenotype was obtained. Second, we provide evidence that the CRISPR/Cas9 system
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mediated gene targeting in heterochromatic regions has no apparent difference compared to that in
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euchromatic regions.
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RESULTS
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Zmzb7 as a marker gene for gene targeting
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To simplify the later mutant screening step, a marker gene Zmzb7 was chosen as our target. Zmzb7 encodes the IspH protein, which is essential for the methyl-D-erythritol-4-phosphate (MEP)
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pathway. Loss of function of Zmzb7 will generate a completely albino plant (Lu et al., 2012). To
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target Zmzb7, we designed a sgRNA for a site located in the 8th exon of the gene. The sgRNA was
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driven by maize U3 promoter, and the Cas9 gene was controlled by a 2 x 35S promoter (Fig. 1A)
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(Feng et al., 2013). The sgRNA-Cas9 expressing plasmid was transformed into maize protoplasts
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to check the mutation efficiency. By RE-PCR-RE (restriction enzyme-PCR-restriction enzyme)
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analysis of the protoplast genomic DNA, a mutated sequence band could be observed easily in the
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agarose gel. The PCR products were then cloned and sent for sequencing. Indel mutations in the
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target site were revealed by the sequencing results (Fig. 1B). Next we transformed the construct
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into maize immature embryos mediated by Agrobacterium. Following the standard maize
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transformation protocol (Frame et al., 2002), we obtained 10 putative transgenic callus events. By
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PCR-RE analysis of the genomic DNA of these events, we determined that the mutation efficiency
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ACCEPTED MANUSCRIPT ranged from 19-31%. PCR products were also sequenced, and Indel mutations were revealed (Fig.
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1C). The putative calli were then used for regeneration. In total about 150 harvested seedlings, one
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plant showed an albino phenotype (Fig. 2B). The albino plant was analyzed by PCR-RE followed
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by PCR product cloning and sequencing. Sequencing results indicated that it was probably a
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mosaic mutant with two mutated alleles, and the ratio of mutational alleles in the whole plant is
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86%. Of the two mutation alleles, one is a 1 bp insertion and the other is a 1 bp deletion, both of
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which will cause frame shift of the protein sequence. Because of the extremely high mutation ratio,
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the plant exhibits an albino phenotype and arrested growth. In addition, we obtained another 2
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putative heterozygous mutant plants or mosaic plants with a mutant allele ratio of 49% and 69%,
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respectively (Fig. 2A-C). These two plants show normal phenotypes (data not shown).
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Sites located in heterochromatic regions could be targeted by the CRISPR/Cas9 system
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The CRISPR/Cas9 system has proved to be robust and efficient for a number of species. In
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Drosophila, it was reported that heterochromatic regions have no effect on CRISPR/Cas9
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mediated genome modification (Yu et al., 2013). In plants, especially maize, which have large
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heterochromatic regions, there is no report of the efficiency of the CRISPR/Cas9 system mediated
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gene targeting in heterochromatic regions. We were interested to address this question. As
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centromere and pericentromere regions usually are thought to be heterochromatic, we decided to
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select genes located in these regions (Dillon and Festenstein, 2002). According to the maize
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genome and transcriptome sequencing data and our anti-CENH3 antibody ChIP-sequencing data
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(Wolfgruber et al., 2009; Fu et al., 2013; He et al., 2013), 12 genes located in the maize
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centromere and pericentromere regions of chromosomes 2 and 5 were selected for targeting
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(Tables 1 and S1). Among these genes, half are actively expressed. On each gene we choose one
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targeting site, and sgRNAs were then designed to target these sites. The plasmids for expressing
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sgRNAs and Cas9 nuclease were mixed and used to transform maize protoplasts and followed by
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PCR-RE analysis. Indel mutations were detected in 5 of these target sites, with an efficiency
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ranging from 2.8% to 27%. Among these 5 target sites, 3 of them were located in active genes and
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2 of them were located in silent genes (Fig. 3). For the other 7 target sites, no mutation was
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detected in 5 sites, and 2 sites were undetermined. Based on these results, we suggest that in maize,
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genes located in heterochromatic regions could be targeted efficiently and independently of
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whether they are expressed.
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Evaluation of off-target events of CRISPR/Cas9 in whole genome
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154 The application of CRISPR/Cas system in human cells showed a high-frequency of off-target
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mutations (Fu et al., 2013; Pattanayak et al., 2013). To assess the off-target possibility of
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CRISPR/Cas9 in maize, the sites similar to target sites were amplified by PCR using transformed
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protoplast DNA as template to perform DNA sequencing. As shown in Table 2, there was no
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mutation in a similar site if there was no NGG (PAM site) (in No. 2) or there were more than 3
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different base pairs (in No. 1). However, if the site was almost identical to the target site
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(including the PAM site) except a single nucleotide (in No. 3), the mutation of 1-bp substitution
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happened at the similar site. In our study, the PAM site was necessary for sgRNA targeting and the
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off-target effects were active at the sites with high similarity to target sites.
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DISCUSSION
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In this study, our results show that the CRISPR/Cas9 system is a robust and efficient tool for
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targeted genome modification in maize. For targeting the Zmzb7 site, transformation experiments
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generated 10 bar-resistant callus events. The mutation ratio in calli is variable, ranging from 19%
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to 31%. We speculate that this may be due to the different T-DNA genomic insertion sites in
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genome region. In some cases, the expression cassettes included in the T-DNA would be silenced.
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From our screening, one seedling showed an expected albino phenotype. The plant also grew very
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slowly. Sequencing results revealed that the plant had mutations with a ratio of 86%, and is most
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probably mosaic mutant with two types of mutant alleles, both of which caused frame shift during
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translation. As callus can be subcultured for a long time, the mutations would be continuously
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occurring. It is possible that if more seedlings are regenerated, the probability to gain a
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homozygous mutant would increase. We also obtained three putative heterozygous mutant plants,
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which should produce homozygous mutants in the next generation. We also designed several
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sgRNAs targeting other genes. By testing the mutation efficiency in protoplasts, we found that
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ACCEPTED MANUSCRIPT only about 50% of sgRNAs could generate mutations successfully (data not shown).This indicates
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that there might be some factors regulating the mutation process. Moreover, the mutation
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efficiency mediated by the CRIPSR/Cas9 system is variable in different plant species (Fauser et al.,
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2014; Zhang et al., 2014). The efficiency is very high in rice, and relatively lower in Arabidopsis.
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Some factors probably affect the efficiency of the CRISPR/Cas9 system, such as gene specificity
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of the target sites, sgRNA sequence, the promoter for Cas9 and sgRNA expression, and T-DNA
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insertion sites (Johnson et al., 2015). Because genome editing process is complicated and there is
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no standard rule for choosing proper target sites, a mutation test in protoplasts or other ways prior
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the transformation is strongly recommended for maize.
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The frequency of obtaining mutant plants and mutation types possibly is related to the target
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gene or target site. Genes not affecting vegetative growth, such as MADS-box genes AP1 (Feng et
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al., 2014), and the gene encoding a noncoding RNA, such as OsPMS3 (Zhang et al., 2014), could
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have less detrimental effects, which would facilitate the recovery of homozygous mutants. The
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mutation types in these genes were mainly small insertions and deletions. Genes encoding key
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enzymes, such as OsEPSPS, had rarely detected mutations of three or more base pairs and
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homozygous mutants, because it would lead to plant death (Zhang et al., 2014). Selecting genes
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with spatial- or temporal-specific expression should be a good strategy. Meiosis-specific genes are
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good candidates. Plants are supposed to be healthy until they proceed to reproductive development
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if the meiosis-specific genes were targeted.
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Our work also indicates that genomic loci in heterochromatic regions in maize could be targeted
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by the CRISPR/Cas9 system. Among the 12 selected sites located in centromere or pericentromere
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regions, 5 sites could be targeted. The others could not or were undetected. The ratio of the sites
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that could be targeted is about 50%. The efficiency shows no difference compared to the sites we
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chose from euchromatic regions. Therefore, we suggest that gene targeting by the CRISPR/Cas9
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system may be independent of the chromatin state of the genes. We also analyzed the expression
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levels of the 12 genes utilizing the published maize transcriptome data, and found that half of
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them are expressed, while the others are not. We checked whether the CRISPR/Cas9 mediated
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gene targeting efficiency is related to the genes’ expression level, and found no correlation.
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the target and similar sites; therefore, the off-target effects are common (Fu et al., 2013;
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Pattanayak et al., 2013). However, off-target effects were reported to appear rarely in rice (Shan et
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al., 2013; Xu et al., 2014; Zhang et al., 2014) and sweet orange (Jia and Wang, 2014). In our study,
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we did not detect off-target effects if the similar site has no PAM motif or has more than two
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mismatches. According to the results of ours and others, off-target mutagenesis is of low concern
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for genetic studies in plants. The best strategy to avoid off-target effects is to identify
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gene-specific sgRNA target sites.
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In summary, our results indicate that the CRISPR/Cas9 system is an efficient tool for targeted
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genome modification in maize in both enchromatic and heterochromatic regions. The fantastic
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system would benefit maize functional genomics study, and ultimately maize breeding.
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MATERIALS AND METHODS
Construction of the sgRNA-Cas9 expression vector
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To target the marker gene Zmzb7 and other sites in maize, we generate plasmids as follows.
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The Cas9 expression cassette in 35S-Cas9-SK was released by digestion with Xma I and Hind
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III (New England Biolabs, UK) and then subcloned into pCambia3301 to produce3301-Cas9
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(Feng et al., 2013). Maize U3 promoter was amplified from B73 genomic DNA using two
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primers (ZmU3-F and ZmU3-R) (Leader, 1994). The sgRNA scaffold was amplified with
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another two primers (sgRNA-F and sgRNA-R) (Feng et al., 2013). Two PCR fragments were
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then used as template to do overlapping PCR to generate ZmU3-sgRNA with primers
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(ZmU3-F and sgRNA-R). PCR products were then cloned into pEasy-Blunt simple vector
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(TransGen Biotech, Beijing, China) to make pU3-sgRNA. Sequence such as 5′-G-N(20)-GG-3′
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or 5′-B-N(21)-GG-3′ in the maize genome region could be chosen as target sites to design
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sgRNA. To make the later mutation analysis simpler, sequences with a restriction enzyme site
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over the Cas9 cutting site would be preferred.
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Maize protoplast transformation
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After finishing the constructs, we first identified the mutation efficiencies in protoplast.
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Maize protoplast transformation is carried out according to previously reported methods with
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some modifications (Zhang et al., 2011). Maize HiII seeds were germinated in the dark at
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30°C for 3 days and then moved to dark conditions at 25°C for another 7 days. Leaves of the
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seedlings would be used to harvest mesophyll protoplasts.
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Leaves were cut into 1mm pieces by new sharp razor blades, and then put into a 100 mL
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triangular flask containing 20 mL enzyme solution (1.5% Cellulase, 0.1% Macerzyme, 0.4
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mol/L Mannitol, 20 mmol/L KCl, 20 mmol/L MES at pH 5.7, 10 mmol/L CaCl2, 0.1% BSA
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and 5 mmol/L β-mercaptoethanol) and vacuum infiltration applied for 30 min (15 Hg),
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followed by 3-4 h shaking (40 r/min) at 25°C in the dark. After digestion, the solution was
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passively flowed through a 40 mm nylon mesh (Millipore, Germany). The protoplasts were
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subjected to centrifugation at 100 g for 3 min. The supernatant was discarded, and then the
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protoplasts were washed with W5 buffer (154 mmol/L NaCl, 5 mmol/L KCl, 125 mmol/L
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CaCl2, 2 mmol/L MES at pH 5.7) two times. The protoplasts were resuspend in Mmg buffer
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(4 mmol/L MES, 0.4 mol/L Mannitol, 15 mmol/L MgCl2 at pH 5.7) at a concentration of
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about 106 cells/mL.
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For PEG mediated transformation, 190 µL protoplast suspension combined with 10 µg
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plasmid (1µg/µL) was added into 2 mL centrifuge tubes. Then 200 µL 40% PEG solution (40%
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PEG4000, 100 mmol/L CaCl2, 0.6 mol/L Mannitol) was added and mixed gently by pipetting.
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The mixture was incubated at 25°C for 18 min. Then, the protoplasts were washed by W5
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solution (1.6 mL) two times, resuspended in W5 solution and incubated in the dark at 25°C
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for 48 h.
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Agrobacterium mediated maize transformation
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Maize HiII seeds were planted in the experimental field in Beijing during May to
ACCEPTED MANUSCRIPT September. The F2 immature zygotic embryos were harvested about 9 days after pollination
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and used for Agrobacterium mediated transformation following previous protocols (Frame et
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al., 2002) with a slight difference. In summary, after embryo dissection, the immature
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embryos were infected by Agrobacterium for 5 min, and then placed on the co-cultivation
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medium at 20°C in the dark for 3 days. After that, embryos were transferred to resting
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medium at 28°C in the dark for 7 days and transferred to selection medium I for 2 weeks with
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the same conditions as on the resting medium. Additionally, they were subcultured on the
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selection medium II for 4-6 weeks. The putative transformed calli were harvested for
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regeneration. The regeneration process includes three two-week sub-culture steps in
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pre-regeneration medium (dark), regeneration medium I (dark) and regeneration medium II
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(light) at 25°C, respectively.
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Mutation analysis of the transformed protoplasts, callus and seedlings
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To detect the mutation efficiency in protoplasts, genomic DNA of the transformed
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protoplasts was extracted using DNA extract kit (Tiangen, Beijing, China). The genomic DNA
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was used as template to perform a PCR reaction. The PCR products were then digested by
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restriction enzymes, and subjected to gel electrophoresis. The gel was imaged by UV and
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photograghed by professional software. The target sites in heterochromatic regions were
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identified by this way. For some target sites, such as Zmzb7 site, mutations cannot be detected
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readily. The genomic DNA was first digested prior to PCR, followed by PCR amplification
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and restriction enzyme digestion. Because the digestion efficiency could not reach 100%, the
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digested wild type genomic DNA would also be amplified by adding 5-10 amplification cycles. To
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detect the mutation efficiency in callus and regenerated seedlings, the protocols are similar to
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those for protoplasts and the genomic DNA was used directly as template for PCR. The PCR
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products were cloned into pMD19-T (Takara, Japan), and usually 30-50 single clones for one
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target site were sent for Sanger sequencing (Ruibio Biotech, Beijing, China).
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ACKNOWLEDGEMENTS
ACCEPTED MANUSCRIPT We thank Prof. Jiankang Zhu for kindly providing the Cas9 expression constructs (Shanghai
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Center for Plant Stress Biology of Chinese Academy of Sciences, China). Prof. Tianyu Wang
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(Institute of Crop Science of Chinese Academy of Agricultural Sciences China) helped us with
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maize transformation. This work was supported by the National Natural Science Foundation of
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China (No. 31320103912).
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SUPPLEMENTARY DATA
307 Table S1. Sequences and restriction enzymes for the target sites.
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Table S2. Primers used in this study.
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CRISPR-Cas system. Nat. Biotechnol. 31:681-683. Li, J.F., Norville, J.E., Aach, J., McCormack, M., Zhang, D.D., Bush, J., Church, G.M., and Sheen, J., 2013b. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat. Biotechnol. 31:688-691.
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Li, X., Dong, L., wang, Z., Zhang, H., Han, C., Liu, B., Wang, X., and Chen, Q., 2014. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 14:327.
Liang, Z., Zhang, K., Chen, K., and Gao, C., 2014. Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. J. Genet. genomics 41:63-68.
Lu, X.M., Hu, X.J., Zhao, Y.Z., Song, W.B., Zhang, M., Chen, Z.L., Chen, W., Dong, Y.B., Wang, Z.H., and Lai, J.S., 2012. Map-based cloning of zb7 encoding an IPP and DMAPP synthase in the MEP pathway of maize. Mol. Plant 5:1100-1112. Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E., and Church, G.M., 2013. RNA-guided human genome engineering via Cas9. Science 339:823-826. Nekrasov, V., Staskawicz, B., Weigel, D., Jones, J.D., and Kamoun, S., 2013. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31:691-693.
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of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.
Nat. Biotechnol. 31:839-843. Perez-Pinera, P., Ousterout, D.G., and Gersbach, C.A., 2012. Advances in targeted genome editing. Curr. Opin. Chem. Biol. 16:268-277. Puchta, H., and Fauser, F., 2013. Gene targeting in plants: 25 years later. Int. J. Dev. Biol. 57:629-637. Shan, Q.W., Wang, Y.P., Li, J., Zhang, Y., Chen, K.L., Liang, Z., Zhang, K., Liu, J.X., Xi, J.J., Qiu,
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J.L., and Gao, C.X., 2013. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31:686-688.
Svitashev, S., Young, J., Schwartz, C., Gao, H., Falco, S.C., and Cigan, A.M., 2015. Targeted mutagenesis, precise gene editing and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiol. pp. 00793.02015.
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Wolfgruber, T.K., Sharma, A., Schneider, K.L., Albert, P.S., Koo, D.H., Shi, J., Gao, Z., Han, F., Lee, H., and Xu, R., 2009. Maize centromere structure and evolution: sequence analysis of centromeres 2 and 5 reveals dynamic loci shaped primarily by retrotransposons. PLoS Genet. 5:e1000743.
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Xu, R., Li, H., Qin, R., Wang, L., Li, L., Wei, P.C., and Yang, J.B. 2014. Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice. Rice 7: 5. Yu, Z., Ren, M., Wang, Z., Zhang, B., Rong, Y.S., Jiao, R., and Gao, G., 2013. Highly efficient genome modifications mediated by CRISPR/Cas9 in Drosophila. Genetics 195:289-291. Zhang, H., Zhang, J., Wei, P., Zhang, B., Gou F., Feng, Z., Mao, Y., Yang, L, Zhang, H., Xu, N., and Zhu, J. 2014. The CRISPR/Cas9 system produces specific and homozygous targete gene editing in rice in one gerneration. Plant Biotech. J. 12:797-807.
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Zhang, Y., Su, J., Duan, S., Ao, Y., Dai, J., Liu, J., Wang, P., Li, Y., Liu, B., Feng, D., Wang, J., and Wang, H.B., 2011. A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes. Plant Methods 7:30.
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Pattanayak, V., Lin, S., Guilinger, J.P., Ma, E., Doudna, J.A., Liu, D.R. 2013. High-throughput
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Figure legend
411 Fig. 1 Detection of CRISPR/Cas9 system mediated mutations on Zmzb7 target site in protoplast
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and callus.
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A: Schematic illustration of the target site and construct for gRNA-Cas9 expression and
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transformation. Black box indicates exons of the Zmzb7 gene. The red arrow shows the target site
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and the red DNA sequence is the target sequence. Underline indicates the Pvu II site. “H” and “X”
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indicates restriction enzyme Hind III and Xma I, respectively. B: RE-PCR-RE results of
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transformed protoplast genomic DNA. Lanes 1 and 2 show PCR products amplified from Pvu II
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digested transformed protoplast genomic DNA and wild type protoplast genomic DNA followed
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by Pvu II digestion, respectively. Lane “WT” shows PCR product amplified from Pvu II digested
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wild type protoplast genomic DNA without the second Pvu II digestion. The right side shows the
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sequencing results of the undigested fragment of lane 1. C: PCR-RE analysis of transgenic callus
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genomic DNA. Lanes 1-10 show PCR amplified from 10 different transgenic callus genomic DNA
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followed by Pvu II digestion. Lane “WT” shows PCR product amplified from wild type genomic
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DNA followed by Pvu II digestion. Mutation efficiencies are shown below the gel picture. The
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below shows the sequencing results which indicating the mutation types in callus. The sequence
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with a red underline is PAM sequence.
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Fig. 2 Detection of CRISPR/Cas9 mediated mutations in regenerated seedlings.
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A: PCR-RE results of transgenic seedlings’ genomic DNA. Lane 1-15 show PCR product
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amplified from genomic DNA of 15 selected transgenic seedlings followed by Pvu II digestion.
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Lanes “WT” shows PCR product amplified from WT genomic DNA followed by Pvu II digestion.
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Mutation efficiencies of partial lanes are shown just below the gel picture. B: Phenotype of
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seedling corresponding to lane 6, which is the expected albino plant. C: Sequencing results of lane
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6, lane 9 and lane 13. The sequence with a red underline is PAM sequence.
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Fig. 3 Detection of CRISPR/Cas9 mediated mutations for target sites in heterochromatic regions.
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Hsg3, Hsg4, Hsg6, Hsg7, Hsg12 were selected target sites with mutations mediated by
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CRISPR/Cas9 system. For all five gel pictures, lanes 1 and 2 repeatedly show PCR products
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Lanes 3 and 4 show PCR products amplified from wild type protoplast genomic DNA followed
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with or without restriction enzyme digestion. The right side shows the sequencing results of the
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undigested fragment of lanes 1 and 2. The sequence with a red underline is PAM sequence.
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Mutation efficiencies are shown below the gel pictures.
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ACCEPTED MANUSCRIPT Table 1. Summary of the target sites in heterochromatic regions selected for targeting by CRISPR/Cas9system Mutation detected by PCR-RE
Gene ID
Hsg1
GRMZM2G091313
Chr.2
5.64
No
Hsg2
GRMZM2G083935
Chr.2
15.99
No
Hsg3
GRMZM2G332562
Chr.5
9.26
Hsg4
GRMZM2G080129
Chr.5
26.24
Hsg5
GRMZM2G170577
Chr.5
4.23
Hsg12
GRMZM2G438243
Chr.2
59.93
Hsg6
GRMZM2G170586
Chr.2
0
Hsg7
GRMZM2G099580
Chr.2
0
Yes
Hsg8
GRMZM2G000411
Chr.5
0
No
Hsg9
GRMZM2G429781
Chr.5
0
No
Hsg10
GRMZM2G135228
Chr.5
0
Undetermined
Hsg11
GRMZM2G342426
Chr.5
0
No
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Chromosome
Relative expression level
Yes Yes No
Yes
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Site Name
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Yes
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Table2. The off-target effects of CRISPR-Cas9 in maize protoplasts
453 No.
Sequence of target site
Sequence of similar site
Mutation
in
similar site ccgcgcgctgcaggcggccatgg
No
2
acaagatgctatgtatcagctgg
tgcagatgctatctatcagcttc
No
3
agaacctgcgaagtgaagatagg
agaacctgcgaagtgaggatagg
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acgagagctgcaggcggccatgg
Yes
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The underlined sequence is PAM. The letters in red are to emphasize the difference.
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S1673-8527(15)00179-4
DOI:
10.1016/j.jgg.2015.10.002
Reference:
JGG 408
To appear in:
Journal of Genetics and Genomics
Received Date: 16 August 2015 Revised Date:
14 October 2015
Accepted Date: 20 October 2015
Please cite this article as: Feng, C., Yuan, J., Wang, R., Liu, Y., Birchler, J.A., Han, F., Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System, Journal of Genetics and Genomics (2015), doi: 10.1016/j.jgg.2015.10.002. 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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Efficient Targeted Genome Modification in Maize Using
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CRISPR/Cas9 System
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Chao Fenga,b,1, Jing Yuana,1, Rui Wanga , Yang Liua,b, James A. Birchlerc and Fangpu
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Hana,*
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a
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Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
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b
University of Chinese Academy of Sciences, Beijing 100049, China
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c
Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400, USA
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1
These authors contributed equally to this work.
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*
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Tel: +86 10 6480 7926, fax: +86 10 6485 4467
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E-mail address: [email protected]
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Correspondending author:
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State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and
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ABSTRACT
31 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system, which is a
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newly developed technology for targeted genome modification, has been successfully used in a
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number of species. In this study, we applied this technology to carry out targeted genome
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modification in maize. A marker gene Zmzb7 was chosen for targeting. The sgRNA-Cas9
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construct was transformed into maize protoplasts, and indel mutations could be detected. A mutant
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seedling with an expected albino phenotype was obtained from screening 120 seedlings generated
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from 10 callus events. Mutation efficiency in maize heterochromatic regions was also investigated.
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Twelve sites with different expression levels in maize centromeres or pericentromere regions were
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selected. The sgRNA-Cas9 constructs were transformed into protoplasts followed by sequencing
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the transformed protoplast genomic DNA. The results show that the genes in heterochromatic
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regions could be targeted by the CRISPR/Cas9 system efficiently, no matter whether they are
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expressed or not. Meanwhile, off-target mutations were not found in the similar sites having no
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PAM motif or having more than two mismatches. Together, our results show that the
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CRISPR/Cas9 system is a robust and efficient tool for genome modification in both euchromatic
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and heterochromatic regions in maize.
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Keywords: CRISPR/Cas9; targeted genome modification; heterochromatic region; maize
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INTRODUCTION
63 As one of the most important crops in the world, maize (Zea mays) is also a model plant for
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genetic research. Traditional strategies for genetic study of maize mostly depend on chemical
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(EMS) treatment or transposon tagging to generate mutant alleles (Candela and Hake, 2008).
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These methods have been proved to be effective, but because the mutations occur randomly, it
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takes time and energy to perform a large screen.
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“Targeted genome modification” is a new concept. The basic principle is to design a nuclease
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that can specifically target a genomic locus, generate a DSB (double strand break) at the site
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(Puchta and Fauser, 2013), and then use the endogenous cellular DSB repair system to modify the
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targeted genomic locus. Two artificial nucleases, ZFN (zinc finger nuclease) and TALEN
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(transcription activator like effector nuclease) were successfully used for targeted genome
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modifications in the past, especially TALENs (Carroll, 2011; Joung and Sander, 2012;
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Perez-Pinera et al., 2012). More recently, a new “targeted genome modification” system, CRISPR
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(clustered regularly interspaced short palindromic repeats)/Cas9 system, was developed and
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quickly spread in the scientific community due to its simple cloning procedure and relatively
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higher efficiency (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013). For the CRISPR/Cas9
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system, the endonuclease Cas9 is guided by a single guide RNA (sgRNA), which includes two
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parts. One of them (usually 20 bp) recognizes the target sites through Watson-Crick base pairing
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between DNA and RNA, and the other forms a structure that recruits the Cas9 endonuclease. Only
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a PAM (NGG) sequence is necessary for target site design, which means almost all genes in a
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genome could be targeted by this system (Hsu et al., 2014). The utilization of CRISPR/Cas9
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system for genome editing has been reported in various species, from bacteria to mammals, which
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supports the point that it hardly has species limits (Bassett an Liu, 2014; Cho et al., 2013; Hwang
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et al., 2013; Jiang et al., 2013; Li et al., 2013a; Li et al., 2013b; Nekrasov et al., 2013; Shan et al.,
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2013). In maize, genome modification by the CRISPR/Cas9 system has been reported previously
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(Liang et al., 2014; Xing Hui-Li, 2014). Mutations mediated by the CRISPR/Cas9 system have
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been detected in maize protoplasts and transgenic seedlings.
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did not work well constitutively. In some cases, the target sites meeting the requirements for
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targeting cannot be targeted (Hwang et al., 2013). The reason is unknown, and the molecular
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mechanisms for Cas9 nuclease to cut the target DNA sequence are to be explored. As the process
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of gene targeting by CRIPSR/Cas9 system depends on the sgRNA to search and target to the
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specific site, it is an interesting question of whether the chromatin environment would influence
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the sgRNA binding ability and change the gene targeting efficiency in plants.
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Here, we report our results of using CRIPSR/Cas9 system to do targeted genome modification
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in maize. First, a marker gene Zmzb7 was successfully mutated, and a mutant plant exhibiting the
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expected phenotype was obtained. Second, we provide evidence that the CRISPR/Cas9 system
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mediated gene targeting in heterochromatic regions has no apparent difference compared to that in
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euchromatic regions.
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RESULTS
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Zmzb7 as a marker gene for gene targeting
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To simplify the later mutant screening step, a marker gene Zmzb7 was chosen as our target. Zmzb7 encodes the IspH protein, which is essential for the methyl-D-erythritol-4-phosphate (MEP)
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pathway. Loss of function of Zmzb7 will generate a completely albino plant (Lu et al., 2012). To
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target Zmzb7, we designed a sgRNA for a site located in the 8th exon of the gene. The sgRNA was
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driven by maize U3 promoter, and the Cas9 gene was controlled by a 2 x 35S promoter (Fig. 1A)
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(Feng et al., 2013). The sgRNA-Cas9 expressing plasmid was transformed into maize protoplasts
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to check the mutation efficiency. By RE-PCR-RE (restriction enzyme-PCR-restriction enzyme)
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analysis of the protoplast genomic DNA, a mutated sequence band could be observed easily in the
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agarose gel. The PCR products were then cloned and sent for sequencing. Indel mutations in the
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target site were revealed by the sequencing results (Fig. 1B). Next we transformed the construct
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into maize immature embryos mediated by Agrobacterium. Following the standard maize
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transformation protocol (Frame et al., 2002), we obtained 10 putative transgenic callus events. By
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PCR-RE analysis of the genomic DNA of these events, we determined that the mutation efficiency
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1C). The putative calli were then used for regeneration. In total about 150 harvested seedlings, one
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plant showed an albino phenotype (Fig. 2B). The albino plant was analyzed by PCR-RE followed
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by PCR product cloning and sequencing. Sequencing results indicated that it was probably a
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mosaic mutant with two mutated alleles, and the ratio of mutational alleles in the whole plant is
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86%. Of the two mutation alleles, one is a 1 bp insertion and the other is a 1 bp deletion, both of
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which will cause frame shift of the protein sequence. Because of the extremely high mutation ratio,
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the plant exhibits an albino phenotype and arrested growth. In addition, we obtained another 2
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putative heterozygous mutant plants or mosaic plants with a mutant allele ratio of 49% and 69%,
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respectively (Fig. 2A-C). These two plants show normal phenotypes (data not shown).
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Sites located in heterochromatic regions could be targeted by the CRISPR/Cas9 system
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The CRISPR/Cas9 system has proved to be robust and efficient for a number of species. In
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Drosophila, it was reported that heterochromatic regions have no effect on CRISPR/Cas9
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mediated genome modification (Yu et al., 2013). In plants, especially maize, which have large
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heterochromatic regions, there is no report of the efficiency of the CRISPR/Cas9 system mediated
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gene targeting in heterochromatic regions. We were interested to address this question. As
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centromere and pericentromere regions usually are thought to be heterochromatic, we decided to
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select genes located in these regions (Dillon and Festenstein, 2002). According to the maize
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genome and transcriptome sequencing data and our anti-CENH3 antibody ChIP-sequencing data
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(Wolfgruber et al., 2009; Fu et al., 2013; He et al., 2013), 12 genes located in the maize
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centromere and pericentromere regions of chromosomes 2 and 5 were selected for targeting
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(Tables 1 and S1). Among these genes, half are actively expressed. On each gene we choose one
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targeting site, and sgRNAs were then designed to target these sites. The plasmids for expressing
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sgRNAs and Cas9 nuclease were mixed and used to transform maize protoplasts and followed by
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PCR-RE analysis. Indel mutations were detected in 5 of these target sites, with an efficiency
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ranging from 2.8% to 27%. Among these 5 target sites, 3 of them were located in active genes and
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2 of them were located in silent genes (Fig. 3). For the other 7 target sites, no mutation was
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detected in 5 sites, and 2 sites were undetermined. Based on these results, we suggest that in maize,
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genes located in heterochromatic regions could be targeted efficiently and independently of
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whether they are expressed.
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Evaluation of off-target events of CRISPR/Cas9 in whole genome
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mutations (Fu et al., 2013; Pattanayak et al., 2013). To assess the off-target possibility of
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CRISPR/Cas9 in maize, the sites similar to target sites were amplified by PCR using transformed
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protoplast DNA as template to perform DNA sequencing. As shown in Table 2, there was no
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mutation in a similar site if there was no NGG (PAM site) (in No. 2) or there were more than 3
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different base pairs (in No. 1). However, if the site was almost identical to the target site
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(including the PAM site) except a single nucleotide (in No. 3), the mutation of 1-bp substitution
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happened at the similar site. In our study, the PAM site was necessary for sgRNA targeting and the
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off-target effects were active at the sites with high similarity to target sites.
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DISCUSSION
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In this study, our results show that the CRISPR/Cas9 system is a robust and efficient tool for
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targeted genome modification in maize. For targeting the Zmzb7 site, transformation experiments
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generated 10 bar-resistant callus events. The mutation ratio in calli is variable, ranging from 19%
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to 31%. We speculate that this may be due to the different T-DNA genomic insertion sites in
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genome region. In some cases, the expression cassettes included in the T-DNA would be silenced.
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From our screening, one seedling showed an expected albino phenotype. The plant also grew very
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slowly. Sequencing results revealed that the plant had mutations with a ratio of 86%, and is most
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probably mosaic mutant with two types of mutant alleles, both of which caused frame shift during
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translation. As callus can be subcultured for a long time, the mutations would be continuously
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occurring. It is possible that if more seedlings are regenerated, the probability to gain a
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homozygous mutant would increase. We also obtained three putative heterozygous mutant plants,
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which should produce homozygous mutants in the next generation. We also designed several
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sgRNAs targeting other genes. By testing the mutation efficiency in protoplasts, we found that
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that there might be some factors regulating the mutation process. Moreover, the mutation
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efficiency mediated by the CRIPSR/Cas9 system is variable in different plant species (Fauser et al.,
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2014; Zhang et al., 2014). The efficiency is very high in rice, and relatively lower in Arabidopsis.
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Some factors probably affect the efficiency of the CRISPR/Cas9 system, such as gene specificity
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of the target sites, sgRNA sequence, the promoter for Cas9 and sgRNA expression, and T-DNA
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insertion sites (Johnson et al., 2015). Because genome editing process is complicated and there is
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no standard rule for choosing proper target sites, a mutation test in protoplasts or other ways prior
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the transformation is strongly recommended for maize.
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The frequency of obtaining mutant plants and mutation types possibly is related to the target
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gene or target site. Genes not affecting vegetative growth, such as MADS-box genes AP1 (Feng et
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al., 2014), and the gene encoding a noncoding RNA, such as OsPMS3 (Zhang et al., 2014), could
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have less detrimental effects, which would facilitate the recovery of homozygous mutants. The
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mutation types in these genes were mainly small insertions and deletions. Genes encoding key
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enzymes, such as OsEPSPS, had rarely detected mutations of three or more base pairs and
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homozygous mutants, because it would lead to plant death (Zhang et al., 2014). Selecting genes
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with spatial- or temporal-specific expression should be a good strategy. Meiosis-specific genes are
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good candidates. Plants are supposed to be healthy until they proceed to reproductive development
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if the meiosis-specific genes were targeted.
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Our work also indicates that genomic loci in heterochromatic regions in maize could be targeted
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by the CRISPR/Cas9 system. Among the 12 selected sites located in centromere or pericentromere
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regions, 5 sites could be targeted. The others could not or were undetected. The ratio of the sites
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that could be targeted is about 50%. The efficiency shows no difference compared to the sites we
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chose from euchromatic regions. Therefore, we suggest that gene targeting by the CRISPR/Cas9
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system may be independent of the chromatin state of the genes. We also analyzed the expression
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levels of the 12 genes utilizing the published maize transcriptome data, and found that half of
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them are expressed, while the others are not. We checked whether the CRISPR/Cas9 mediated
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gene targeting efficiency is related to the genes’ expression level, and found no correlation.
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the target and similar sites; therefore, the off-target effects are common (Fu et al., 2013;
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Pattanayak et al., 2013). However, off-target effects were reported to appear rarely in rice (Shan et
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al., 2013; Xu et al., 2014; Zhang et al., 2014) and sweet orange (Jia and Wang, 2014). In our study,
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we did not detect off-target effects if the similar site has no PAM motif or has more than two
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mismatches. According to the results of ours and others, off-target mutagenesis is of low concern
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for genetic studies in plants. The best strategy to avoid off-target effects is to identify
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gene-specific sgRNA target sites.
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In summary, our results indicate that the CRISPR/Cas9 system is an efficient tool for targeted
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genome modification in maize in both enchromatic and heterochromatic regions. The fantastic
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system would benefit maize functional genomics study, and ultimately maize breeding.
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MATERIALS AND METHODS
Construction of the sgRNA-Cas9 expression vector
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To target the marker gene Zmzb7 and other sites in maize, we generate plasmids as follows.
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The Cas9 expression cassette in 35S-Cas9-SK was released by digestion with Xma I and Hind
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III (New England Biolabs, UK) and then subcloned into pCambia3301 to produce3301-Cas9
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(Feng et al., 2013). Maize U3 promoter was amplified from B73 genomic DNA using two
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primers (ZmU3-F and ZmU3-R) (Leader, 1994). The sgRNA scaffold was amplified with
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another two primers (sgRNA-F and sgRNA-R) (Feng et al., 2013). Two PCR fragments were
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then used as template to do overlapping PCR to generate ZmU3-sgRNA with primers
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(ZmU3-F and sgRNA-R). PCR products were then cloned into pEasy-Blunt simple vector
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(TransGen Biotech, Beijing, China) to make pU3-sgRNA. Sequence such as 5′-G-N(20)-GG-3′
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or 5′-B-N(21)-GG-3′ in the maize genome region could be chosen as target sites to design
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sgRNA. To make the later mutation analysis simpler, sequences with a restriction enzyme site
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over the Cas9 cutting site would be preferred.
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Maize protoplast transformation
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After finishing the constructs, we first identified the mutation efficiencies in protoplast.
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Maize protoplast transformation is carried out according to previously reported methods with
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some modifications (Zhang et al., 2011). Maize HiII seeds were germinated in the dark at
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30°C for 3 days and then moved to dark conditions at 25°C for another 7 days. Leaves of the
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seedlings would be used to harvest mesophyll protoplasts.
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Leaves were cut into 1mm pieces by new sharp razor blades, and then put into a 100 mL
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triangular flask containing 20 mL enzyme solution (1.5% Cellulase, 0.1% Macerzyme, 0.4
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mol/L Mannitol, 20 mmol/L KCl, 20 mmol/L MES at pH 5.7, 10 mmol/L CaCl2, 0.1% BSA
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and 5 mmol/L β-mercaptoethanol) and vacuum infiltration applied for 30 min (15 Hg),
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followed by 3-4 h shaking (40 r/min) at 25°C in the dark. After digestion, the solution was
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passively flowed through a 40 mm nylon mesh (Millipore, Germany). The protoplasts were
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subjected to centrifugation at 100 g for 3 min. The supernatant was discarded, and then the
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protoplasts were washed with W5 buffer (154 mmol/L NaCl, 5 mmol/L KCl, 125 mmol/L
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CaCl2, 2 mmol/L MES at pH 5.7) two times. The protoplasts were resuspend in Mmg buffer
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(4 mmol/L MES, 0.4 mol/L Mannitol, 15 mmol/L MgCl2 at pH 5.7) at a concentration of
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about 106 cells/mL.
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For PEG mediated transformation, 190 µL protoplast suspension combined with 10 µg
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plasmid (1µg/µL) was added into 2 mL centrifuge tubes. Then 200 µL 40% PEG solution (40%
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PEG4000, 100 mmol/L CaCl2, 0.6 mol/L Mannitol) was added and mixed gently by pipetting.
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The mixture was incubated at 25°C for 18 min. Then, the protoplasts were washed by W5
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solution (1.6 mL) two times, resuspended in W5 solution and incubated in the dark at 25°C
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for 48 h.
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Agrobacterium mediated maize transformation
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Maize HiII seeds were planted in the experimental field in Beijing during May to
ACCEPTED MANUSCRIPT September. The F2 immature zygotic embryos were harvested about 9 days after pollination
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and used for Agrobacterium mediated transformation following previous protocols (Frame et
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al., 2002) with a slight difference. In summary, after embryo dissection, the immature
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embryos were infected by Agrobacterium for 5 min, and then placed on the co-cultivation
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medium at 20°C in the dark for 3 days. After that, embryos were transferred to resting
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medium at 28°C in the dark for 7 days and transferred to selection medium I for 2 weeks with
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the same conditions as on the resting medium. Additionally, they were subcultured on the
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selection medium II for 4-6 weeks. The putative transformed calli were harvested for
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regeneration. The regeneration process includes three two-week sub-culture steps in
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pre-regeneration medium (dark), regeneration medium I (dark) and regeneration medium II
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(light) at 25°C, respectively.
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Mutation analysis of the transformed protoplasts, callus and seedlings
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To detect the mutation efficiency in protoplasts, genomic DNA of the transformed
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protoplasts was extracted using DNA extract kit (Tiangen, Beijing, China). The genomic DNA
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was used as template to perform a PCR reaction. The PCR products were then digested by
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restriction enzymes, and subjected to gel electrophoresis. The gel was imaged by UV and
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photograghed by professional software. The target sites in heterochromatic regions were
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identified by this way. For some target sites, such as Zmzb7 site, mutations cannot be detected
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readily. The genomic DNA was first digested prior to PCR, followed by PCR amplification
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and restriction enzyme digestion. Because the digestion efficiency could not reach 100%, the
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digested wild type genomic DNA would also be amplified by adding 5-10 amplification cycles. To
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detect the mutation efficiency in callus and regenerated seedlings, the protocols are similar to
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those for protoplasts and the genomic DNA was used directly as template for PCR. The PCR
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products were cloned into pMD19-T (Takara, Japan), and usually 30-50 single clones for one
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target site were sent for Sanger sequencing (Ruibio Biotech, Beijing, China).
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ACKNOWLEDGEMENTS
ACCEPTED MANUSCRIPT We thank Prof. Jiankang Zhu for kindly providing the Cas9 expression constructs (Shanghai
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Center for Plant Stress Biology of Chinese Academy of Sciences, China). Prof. Tianyu Wang
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(Institute of Crop Science of Chinese Academy of Agricultural Sciences China) helped us with
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maize transformation. This work was supported by the National Natural Science Foundation of
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China (No. 31320103912).
305 306
SUPPLEMENTARY DATA
307 Table S1. Sequences and restriction enzymes for the target sites.
309
Table S2. Primers used in this study.
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Pattanayak, V., Lin, S., Guilinger, J.P., Ma, E., Doudna, J.A., Liu, D.R. 2013. High-throughput
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Figure legend
411 Fig. 1 Detection of CRISPR/Cas9 system mediated mutations on Zmzb7 target site in protoplast
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and callus.
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A: Schematic illustration of the target site and construct for gRNA-Cas9 expression and
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transformation. Black box indicates exons of the Zmzb7 gene. The red arrow shows the target site
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and the red DNA sequence is the target sequence. Underline indicates the Pvu II site. “H” and “X”
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indicates restriction enzyme Hind III and Xma I, respectively. B: RE-PCR-RE results of
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transformed protoplast genomic DNA. Lanes 1 and 2 show PCR products amplified from Pvu II
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digested transformed protoplast genomic DNA and wild type protoplast genomic DNA followed
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by Pvu II digestion, respectively. Lane “WT” shows PCR product amplified from Pvu II digested
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wild type protoplast genomic DNA without the second Pvu II digestion. The right side shows the
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sequencing results of the undigested fragment of lane 1. C: PCR-RE analysis of transgenic callus
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genomic DNA. Lanes 1-10 show PCR amplified from 10 different transgenic callus genomic DNA
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followed by Pvu II digestion. Lane “WT” shows PCR product amplified from wild type genomic
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DNA followed by Pvu II digestion. Mutation efficiencies are shown below the gel picture. The
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below shows the sequencing results which indicating the mutation types in callus. The sequence
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with a red underline is PAM sequence.
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Fig. 2 Detection of CRISPR/Cas9 mediated mutations in regenerated seedlings.
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A: PCR-RE results of transgenic seedlings’ genomic DNA. Lane 1-15 show PCR product
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amplified from genomic DNA of 15 selected transgenic seedlings followed by Pvu II digestion.
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Lanes “WT” shows PCR product amplified from WT genomic DNA followed by Pvu II digestion.
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Mutation efficiencies of partial lanes are shown just below the gel picture. B: Phenotype of
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seedling corresponding to lane 6, which is the expected albino plant. C: Sequencing results of lane
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6, lane 9 and lane 13. The sequence with a red underline is PAM sequence.
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436 437
Fig. 3 Detection of CRISPR/Cas9 mediated mutations for target sites in heterochromatic regions.
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Hsg3, Hsg4, Hsg6, Hsg7, Hsg12 were selected target sites with mutations mediated by
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CRISPR/Cas9 system. For all five gel pictures, lanes 1 and 2 repeatedly show PCR products
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Lanes 3 and 4 show PCR products amplified from wild type protoplast genomic DNA followed
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with or without restriction enzyme digestion. The right side shows the sequencing results of the
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undigested fragment of lanes 1 and 2. The sequence with a red underline is PAM sequence.
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Mutation efficiencies are shown below the gel pictures.
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ACCEPTED MANUSCRIPT Table 1. Summary of the target sites in heterochromatic regions selected for targeting by CRISPR/Cas9system Mutation detected by PCR-RE
Gene ID
Hsg1
GRMZM2G091313
Chr.2
5.64
No
Hsg2
GRMZM2G083935
Chr.2
15.99
No
Hsg3
GRMZM2G332562
Chr.5
9.26
Hsg4
GRMZM2G080129
Chr.5
26.24
Hsg5
GRMZM2G170577
Chr.5
4.23
Hsg12
GRMZM2G438243
Chr.2
59.93
Hsg6
GRMZM2G170586
Chr.2
0
Hsg7
GRMZM2G099580
Chr.2
0
Yes
Hsg8
GRMZM2G000411
Chr.5
0
No
Hsg9
GRMZM2G429781
Chr.5
0
No
Hsg10
GRMZM2G135228
Chr.5
0
Undetermined
Hsg11
GRMZM2G342426
Chr.5
0
No
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Chromosome
Relative expression level
Yes Yes No
Yes
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Site Name
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Yes
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Table2. The off-target effects of CRISPR-Cas9 in maize protoplasts
453 No.
Sequence of target site
Sequence of similar site
Mutation
in
similar site ccgcgcgctgcaggcggccatgg
No
2
acaagatgctatgtatcagctgg
tgcagatgctatctatcagcttc
No
3
agaacctgcgaagtgaagatagg
agaacctgcgaagtgaggatagg
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acgagagctgcaggcggccatgg
Yes
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The underlined sequence is PAM. The letters in red are to emphasize the difference.
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