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Targeted Gene Mutation in Rice Using a CRISPR-Cas9 System
采用CRISPR-Cas9 系统进行水稻基因定点突变   

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Molecular Plant
Nov 2013


RNA-guided genome editing (RGE) using bacterial type II cluster regularly interspaced short palindromic repeats (CRISPR)–associated nuclease (Cas) has emerged as a simple and versatile tool for genome editing in many organisms including plant and crop species. In RGE based on the Streptococcus pyogenes CRISPR-Cas9 system, the Cas9 nuclease is directed by a short single guide RNA (gRNA or sgRNA) to generate double-strand breaks (DSB) at the specific sites of chromosomal DNA, thereby introducing mutations at the DSB by error-prone non-homologous end joining repairing. Cas9-gRNA recognizes targeted DNA based on complementarity between a gRNA spacer (~ 20 nt long leading sequence of gRNA) and its targeted DNA which precedes a protospacer-adjacent motif (PAM, Figure 1). In this protocol, we describe the general procedures for plant RGE using CRISPR-Cas9 system and Agrobacterium-mediated transformation. The protocol includes gRNA design, Cas9-gRNA plasmid construction and mutation detection (genotyping) for rice RGE and could be adapted for other plant species.

Figure 1. Schematic illustration of CRISPR-Cas9 system

Materials and Reagents

  1. Oryza sativa L. ssp japonica, Kitaake, Nipponbare, or other cultivars
  2. Agrobacterium tumefaciens strain EHA105
  3. pRGEB31 (Xie and Yang, 2013) (Addgene plasmid 51295 , plasmid map is shown in Figure 2)

    Figure 2. Schematic illustration of pRGE31 and pRGEB31 vectors. The pRGEB31 vector was used in this protocol.

  4. Bsa I (New England Biolabs, catalog number: R0535S )
  5. 70%, 100% ethanol
  6. Alkaline phosphatase, calf intestinal (CIP) (New England Biolabs, catalog number: M0290S )
  7. T4 DNA ligase (New England Biolabs, catalog number: M0202S )
  8. T4 polynucleotide kinase (T4 PNK) (New England Biolabs, catalog number: M0201S )
  9. T7 endonuclease I (T7EI) (New England Biolabs, catalog number: M0302S )
  10. GoTaq DNA polymerase (Promega Corporation, catalog number: M3001 )
  11. Phusion high-fidelity polymerase (Thermo Fisher Scientific, catalog number: F530S )
  12. 5x green GoTaq® reaction buffer (Promega Corporation, catalog number: M7911 )
  13. QIAGEN plasmid mini kit (QIAGEN, catalog number: 12123 )
  14. QIAquick PCR purification kit (QIAGEN, catalog number: 28104 )
  15. Hexadecyltrimethylammonium bromide (CTAB) (Sigma-Aldrich, catalog number: H9151 )
  16. Sodium lauroyl sarcosinate (sarkosyl) (Thermo Fisher Scientific, catalog number: BP235-500 )
  17. CTAB buffer (see Recipes)


  1. 37 °C water bath
  2. Thermal cycler
  3.  DNA electrophoresis apparatus
  4.  Microcentrifuge


  1. Design gRNAs to target the genes of interest
    1. Choose targeted sites and design gRNA spacer sequence as illustrated in Figure 1. For eight model plant species, specific gRNA spacers could be readily designed using the CRISPR-PLANT database (Xie et al., 2014) (http://www.genome.arizona.edu/crispr). Following factors may be considered for designing gRNAs.
      1. To functionally knock-out the genes of interest, designed gRNAs should introduce DSB close to 5’-end of coding region or located in the essential domains for genes’ function.  
      2. Selected gRNA spacers/target sequence should have sufficient specificity (we recommend that gRNA spacers are ranked with class 0.0 and 1.0 specificity in CRISPR-PLANT database) to avoid off-target editing.
      3. (Optional) Check gRNA folding, gRNA with three stem-loops as Figure 1 are preferred.
      4. (Optional) If possible, design a gRNA which would generate mutations at a restriction nuclease enzyme (RE) site in order to introduce RFLP (restriction fragment length polymorphism) for genotyping.
    2. After selection of targeted sites and spacer sequences, DNA oligos are synthesized to construct the gRNAs as described in Figure 3. Appropriate adaptor sequences should be added for cloning. The reverse primer should contain an adaptor of 5’-AAAC-3’ whereas the forward primer should include an adaptor with 5’-GGCA-3’ (see Figure 3 Case 1, gRNA-spacer starts with A) or 5’-GGC-3’ (Figure 3 Case 2, gRNA-spacer starts with G/C/T).
      Note: The gRNAs designed as cases 1 and 2 in Figure 3 showed comparable efficiency in our experiments.

      Figure 3. A simple guide for designing DNA oligos to construct the RGE plasmid (Xie et al., 2014)

  2. Construct gRNA-Cas9 plasmid
    1. Digest the pRGEB31 vector (see Figure 3 for vector details) by Bsa I.

      2 µg
      10x NEB Buffer 4
      2 µl
      10x BSA
      2 µl
      Bsa I (NEB)
      1 µl
      Add H2O to
      20 µl

    2. Incubate at 37 °C for 2-4 h.
    3. (Optional) Add 0.5 µl of CIP to dephosphorylate the vector by incubating at 37 °C for 30 min.
    4. Purify the the digested vector using the QIAquick PCR purification kit.
    5. Prepare DNA oligo-duplex in a PCR tube as following:

      Forward oligo (100 uM)
      1 µl
      Reverse oligo (100 uM)
      1 µl
      10x T4 DNA ligase Buffer
      1 µl
      T4 PNK (NEB)
      0.5 µl
      6.5 µl

      Note: T4 PNK is not required if the vector was not treated with CIP in step 5.

    6. Incubate the tube in a thermal cycler using the following program.

      37 °C
      60 min
      95 °C
      10 min
      Cool down to 25 °C at 0.1 °C/sec

    7. Make a 1: 200 dilution of oligo-duplex for ligation.
    8. Ligate the diluted oligo-duplex into vector:

      Bsa I digested vector
      n µl (~50 ng)
      Oligo-duplex (diluted)
      1 µl
      10x T4 DNA ligase Buffer
      0.5 µl
      T4 ligase (NEB)
      1 µl
      Add H2O to
      5 µl

    9. Incubate at room temperature (25 °C) for 2-4 h or at 4 °C overnight.
    10. Transform E. coli DH5α competent cells (homemade) using 1 µl of ligation product.
    11. Inoculate 2-4 colonies in LB medium with 50 µg/ml kanamycin.
    12.  Purify plasmids from the transformed DH5α cells using QIAGEN Plasmid Mini kit.
    13. Confirm pRGEB31-gRNA plasmid constructs by Sanger sequencing using M13R (-48) primer.

  3. Rice transformation
    1. Introduce the pRGEB31-gRNA plasmid into Agrobacterium strain EHA105.
    2. Transform rice embryogenic callus using standard Agrobacterium-mediated transformation method

  4. Genotyping
    Note: The Cas9-gRNA introduced mutation could be examined in hygromycin B selected calli or transgenic plants. To examine Cas9-gRNA introduced mutations, a pair of gene specific primers is required to amplify the targeted region. Because of the high efficiency of Cas9-gRNA system in rice, steps 22 and 23 are optional and the mutation at targeting site could be readily examined by direct Sanger sequencing of PCR product (step 23).
    1. Genomic DNA extraction.
      1. Ground 10-50 mg of leaves/calli of transgenic rice plants in a 1.5 ml-tube.
      2. Add 200 µl of heated (60 °C) CTAB buffer to the tube, mixed immediately and incubate at 60 °C for 10 min with occassional shaking.
      3. Add 80 µl of chloroform, vortex and incubate in an end-to-top rocker at room temperature for 20 min.
      4. Centrifuge at 14,000 x g for 5 min at room temperature.
      5. Transfer upper aqueous phase to a fresh tube, add 1/10 Vol of sodium acetate (3 M, pH 5.3) and 2.5 Vol of 100% ethanol, and incubate on ice for 10 min.
      6. Centrifuge at 14,000 x g for 5 min at room temperature.
      7. Discard the liquid.
      8. Add 0.5 ml of 70% ethanol and centrifuge at 14,000 x g for 5min.
      9. Discard the supernatant liquid and dry the pellet in air.
      10. Dissolve the genomic DNA in 50-100 µl of H2O.
    2. Setup reactions for PCR amplification using genomic DNAs extracted from transgenic rice and wild type plant.

      Rice genomic DNA
      100 ng
      dNTP (10 mM)
      1 µl
      Forward primer (10 μM)
      1 µl
      Reverse primer (10 µM)
      1 µl
      5x Green GoTaq® Reaction Buffer
      10 µl
      Taq polymerase (2 U/µl)
      1 µl
      Add sterile distilled H2O to
      50 µl

    3. Amplify the targeted region by PCR [annealing temperature (x) and extension time (n) depend on primers].

      95 °C
      3 min
      1 cycle
      95 °C
      15 sec
      35 cycles
      x °C
      20 sec

      72 °C
      n min

      72 °C
      2 min
      1 cycle
      4 °C
      1 cycle

    4. (Optional) If RFLP is introduced (examples was shown in Figure 4)
      1. Add 5 U of appreciate restriction enzyme to 25 µl of PCR product (~200 ng).
      2. Incubate at 37 °C for 2-4 h.
      3. Analyze the digested DNA fragment by 1% agarose gel electrophoresis.
    5. (Optional) If no RFLP was introduced, use T7E1 assay to detect mutations:
      Note: High fidelity DNA polymerase (Phusion DNA polymerase) should be used in step 19 for T7E1 assay. T7E1 assay could not detect homozygous mutation. Examples (line #1 and #3) were shown in Figure 4.
      1. Purify the PCR products using PCR purification column.
      2. Set up the T7E1 assay in PCR tubes as following:

        PCR product
        100-200 ng
        10x NEB buffer 2
        1 µl
        Add H2O to
        9.5 µl

      3. Denature and anneal PCR products using the following program:

        95 °C
        10 min
        Ramp to 85 °C at 0.1 °C/sec

        85 °C
        5 min
        Ramp to 65 °C at 0.1 °C/sec

        65 °C
        2 min
        Ramp to 45 °C at 0.1 °C/sec

        45 °C
        2 min
        Ramp to 25 °C at 0.1 °C/sec

        25 °C

      4. Add 0.5 µl of T7E1 to the annealed PCR product.
      5. Incubate the reaction at 37 °C for 1 h.
      6. Analyze the digestion by agarose gel electrophoresis.
    6. Clone and sequence the PCR product (at least 4 colonies) to confirm the mutation in transgenic plants.
      Note: T7E1 enzyme is not absolutely mismatch specific; it also nicks dsDNA slowly. Therefore, it is important to control the enzyme amount and reaction time and carefully interpret the results in T7EI assay.

      Figure 4. Examples of PCR-RFLP assay (A) or T7E1 assay (B) using T0 transgenic plants. In PCR-RFLP assay, mutated DNA is resistant to RE digestion whereas wild type DNA would be cut into pieces. No wild type DNA was detected in transgenic lines #1, #3, and #4 (T0 generation) based on PCR-RFLP results, suggesting highly efficient mutation rate of RGE in rice. Of note, the size of extra band from line #1 and #3 in the PCR-T7E1 assay was different from the expected size in line #4, which was likely resulting from imperfect specificity of T7E1 enzyme. Based on these assays and final sanger sequencing results, lines #1 and #3 were found to contain homozygous, identical mutations at the targeted site whereas line #4 contained heterozygous, different mutations at the targeted site.


  1. CTAB buffer (100 ml)
    Dissolve 2.56 g of sorbitol, 1 g of Sarkosyl and 0.8 g of CTAB in 60 ml H2O.
    Add 11 ml of Tris buffer (pH 8.0, 1 M), 4.4 ml of EDTA (pH 8.0) and 16 ml of NaCl (5 M)
    Heat the solution briefly to dissolve CTAB and Sarkosyl
    Add dH2O to 100 ml
    Autoclave and stored at room temperature


This work was supported by Pennsylvania State University and a research grant from NSF Plant Genome Research Program (DBI-0922747) to YY.


  1. Xie, K. and Yang, Y. (2013). RNA-guided genome editing in plants using a CRISPR-Cas system. Mol Plant 6(6): 1975-1983.
  2. Xie, K., Zhang, J. and Yang, Y. (2014). Genome-wide prediction of highly specific guide RNA spacers for the CRISPR-Cas9 mediated genome editing in model plants and major crops. Mol Plant 7(5): 923-926.


使用细菌II型簇定期间隔的短回文重复序列(CRISPR)相关核酸酶(Cas)的RNA指导的基因组编辑(RGE)已经作为用于在包括植物和作物物种的许多生物体中的基因组编辑的简单和通用工具而出现。在基于化脓性链球菌CRISPR-Cas9系统的RGE中,Cas9核酸酶由短的单引导RNA(gRNA或sgRNA)引导以在染色体的特定位点产生双链断裂(DSB) DNA,从而通过易错的非同源末端连接修复在DSB处引入突变。 Cas9-gRNA基于gRNA间隔区(约20nt的gRNA的前导序列)与其在原间质体相邻基序(PAM,图1)之前的靶DNA之间的互补性识别靶向DNA。在该协议中,我们描述了使用CRISPR-Cas9系统和农杆菌介导的转化的植物RGE的一般程序。该协议包括gRNA设计,Cas9-gRNA质粒构建和水稻RGE的突变检测(基因分型),可适用于其他植物物种。

图1. CRISPR-Cas9系统示意图


  1. 水稻 L.ssp japonica ,Kitaake,Nipponbare或其他栽培品种
  2. 根癌农杆菌菌株EHA105
  3. pRGEB31(Xie和Yang,2013)(Addgene质粒51295,质粒图谱显示于图2)

    图2. pRGE31和pRGEB31载体的示意图。在此协议中使用pRGEB31载体。

  4. I(New England Biolabs,目录号:R0535S)
  5. 70%,100%乙醇
  6. 碱性磷酸酶,小牛肠(CIP)(New England Biolabs,目录号:M0290S)
  7. T4 DNA连接酶(New England Biolabs,目录号:M0202S)
  8. T4多核苷酸激酶(T4 PNK)(New England Biolabs,目录号:M0201S)
  9. T7核酸内切酶I(T7EI)(New England Biolabs,目录号:M0302S)
  10. GoTaq DNA聚合酶(Promega Corporation,目录号:M3001)
  11. Phusion高保真聚合酶(Thermo Fisher Scientific,目录号:F530S)
  12. 5x绿色GoTaq反应缓冲液(Promega Corporation,目录号:M7911)
  13. QIAGEN质粒小试剂盒(QIAGEN,目录号:12123)
  14. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28104)
  15. 十六烷基三甲基溴化铵(CTAB)(Sigma-Aldrich,目录号:H9151)
  16. 月桂酰肌氨酸钠(sarkosyl)(Thermo Fisher Scientific,目录号:BP235-500)
  17. CTAB缓冲区(请参阅配方)


  1. 37°C水浴
  2. 热循环仪
  3.   DNA电泳仪
  4.  微量离心机


  1. 设计gRNA以靶向感兴趣的基因
    1. 选择靶向位点并设计如图1所示的gRNA间隔序列。对于8种模型植物物种,可以使用CRISPR-PLANT数据库容易地设计特异性gRNA间隔物(Xie等人,2014)(< a target ="_ blank"href ="http://www.genome.arizona.edu/crispr"> http://www.genome.arizona.edu/crispr )。设计gRNA时可考虑以下因素。
      1. 为了功能性敲除感兴趣的基因,设计的gRNA应将DSB引入靠近编码区5'-末端或位于基因功能的基本结构域中。  
      2. 选择的gRNA间隔区/靶序列应具有足够的特异性(我们建议gRNA间隔区在CRISPR-PLANT数据库中按0.0和1.0特异性排序)以避免 脱离目标编辑
      3. (可选)检查gRNA折叠,优选具有三个茎环的gRNA,如图1所示
      4. (可选)如果可能,设计一个gRNA,其将在限制性核酸酶(RE)位点产生突变,以引入用于基因分型的RFLP(限制性片段长度多态性)。
    2. 在选择靶位点和间隔序列之后,合成DNA寡核苷酸以构建如图3所述的gRNA。应当添加合适的接头序列用于克隆。反向引物应当包含5'-AAAC-3'的接头,而正向引物应该包括具有5'-GGCA-3'的接头(参见图3情况1,gRNA-间隔物以A开始)或5'-GGC -3'(图3情况2,gRNA-间隔物以G/C/T开始) 注意:图3中设计为病例1和2的gRNA在我们的实验中显示出相当的效率。

      图3.设计DNA寡核苷酸以构建RGE质粒的简单指南(Xie et al。,2014)

  2. 构建gRNA-Cas9质粒
    1. 通过 I,摘要pRGEB31载体(见图3的载体细节)。

      10x NEB缓冲区4
      10x BSA
      Bsa I(NEB)
      将H 2 O添加到

    2. 在37℃孵育2-4小时
    3. (可选)加入0.5μlCIP,通过在37℃孵育30分钟使载体去磷酸化。
    4. 使用QIAquick PCR纯化试剂盒纯化消化的载体
    5. 在PCR管中制备DNA寡核苷酸,如下:

      10×T4 DNA连接酶缓冲液
      T4 PNK(NEB)
      H sub 2 O

      注意:如果在步骤5中不用CIP处理载体,则不需要T4 PNK。
    6. 使用以下程序在热循环仪中孵育试管。


    7. 制备1:20稀释的寡聚双链体用于连接
    8. 将稀释的寡聚双链体导入载体:

      n μl(〜50 ng)
      10×T4 DNA连接酶缓冲液
      将H 2 O添加到

    9. 在室温(25℃)孵育2-4小时或在4℃过夜
    10. 转换 E。 大肠杆菌DH5α感受态细胞(自制),使用1μl连接产物
    11. 在含有50μg/ml卡那霉素的LB培养基中接种2-4个菌落
    12.  使用QIAGEN Plasmid Mini试剂盒从转化的DH5α细胞中纯化质粒
    13. 使用M13R(-48)引物通过Sanger测序确认pRGEB31-gRNA质粒构建体。

  3. 水稻转化
    1. 将pRGEB31-gRNA质粒导入农杆菌菌株EHA105中。
    2. 使用标准土壤杆菌介导的转化方法转化水稻胚胎发生的愈伤组织

  4. 基因分型
    注意:可以在潮霉素B选择的愈伤组织或转基因植物中检查Cas9-gRNA引入的突变。 为了检查Cas9-gRNA导入的突变,需要一对基因特异性引物来扩增靶向区域。 由于水稻中Cas9-gRNA系统的高效率,步骤22和23是任选的,并且可以通过PCR产物的直接Sanger测序容易地检查靶向位点的突变(步骤23)。
    1. 基因组DNA提取。
      1. 在1.5ml管中研磨10-50mg转基因稻植物的叶/愈伤组织
      2. 向管中加入200μl加热(60℃)CTAB缓冲液,立即混合并在60℃下伴随振荡孵育10分钟。
      3. 加入80μl氯仿,涡旋并在端到端摇臂中在室温下孵育20分钟
      4. 在室温下以14,000×g离心5分钟
      5. 将上层水相转移至新鲜管中,加入1/10体积的乙酸钠(3M,pH5.3)和2.5体积的100%乙醇,并在冰上孵育10分钟。
      6. 在室温下以14,000×g离心5分钟
      7. 丢弃液体。
      8. 加入0.5ml的70%乙醇,并以14,000×g离心5分钟
      9. 弃去上清液,在空气中干燥沉淀
      10. 将基因组DNA溶解在50-100μlH 2 O中。
    2. 使用从转基因水稻和野生型植物提取的基因组DNA进行PCR扩增的设置反应
      100 ng
      dNTP(10mM) 1微升
      5x Green GoTaq ®反应缓冲液
      Taq聚合酶(2U /μl) 1微升
      将无菌蒸馏的H 2 O加入到

    3. 通过PCR扩增靶区域[退火温度(x)和延伸时间(n)取决于引物]
      x °C



    4. (可选)如果引入RFLP(示例如图4所示)
      1. 向25μlPCR产物(〜200ng)中加入5U明胶酶限制酶
      2. 在37℃孵育2-4小时
      3. 通过1%琼脂糖凝胶电泳分析消化的DNA片段
    5. (可选)如果没有引入RFLP,使用T7E1测定法检测突变:
      注意:在T7E1测定的步骤19中应使用高保真DNA聚合酶(Phusion DNA聚合酶)。 T7E1测定不能检测纯合突变。 示例(行#1和#3)如图4所示。
      1. 使用PCR纯化柱纯化PCR产物
      2. 在PCR管中设置T7E1测定如下:

        100-200 ng
        10x NEB缓冲区2
        将H 2 O添加到

      3. 使用以下程序使PCR产物变性和退火:


      4. 使用以下程序使PCR产物变性和退火:

        Ramp to 65 °C at 0.1 °C/sec

        65 °C
        2 min
        Ramp to 45 °C at 0.1 °C/sec


      5. 向退火的PCR产物中加入0.5μlT7E1。
      6. 将反应在37℃下孵育1小时
      7. 通过琼脂糖凝胶电泳分析消化
    6. 克隆和序列PCR产物(至少4个菌落)以确认转基因植物中的突变。



  1. CTAB缓冲液(100ml) 将2.56g山梨醇,1g Sarkosyl和0.8g CTAB溶解在60ml H 2 O中。
    加入11ml Tris缓冲液(pH8.0,1M),4.4ml EDTA(pH8.0)和16ml NaCl(5M)
    将dH <2> O添加到100 ml




  1. Xie,K.and Yang,Y。(2013)。 使用CRISPR-Cas系统在植物中进行RNA指导的基因组编辑 Mol Plant 6(6):1975-1983。
  2. Xie,K.,Zhang,J.and Yang,Y.(2014)。 在模型植物中CRISPR-Cas9介导的基因组编辑的高度特异性引导RNA间隔区的全基因组预测 和主要作物。 Mol Plant 7(5):923-926。
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引用:Xie, K., Minkenberg, B. and Yang, Y. (2014). Targeted Gene Mutation in Rice Using a CRISPR-Cas9 System. Bio-protocol 4(17): e1225. DOI: 10.21769/BioProtoc.1225.