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Multiplex Gene Editing via CRISPR/Cas9 System in Sheep
通过CRISPR/Cas9系统在绵羊中进行多重基因编辑   

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Abstract

Sheep is a major large animal model for studying development and disease in biomedical research. We utilized CRISPR/Cas9 system successfully to modify multiple genes in sheep. Here we provide a detailed protocol for one-cell-stage embryo manipulation by co-injecting Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and BCO2) to create genetic-modified sheep. Procedure described sgRNA design, construction of gRNA-Cas9 plasmid, efficient detection in fibroblast, embryos and sheep, and some manipulative technologies. Our findings suggested that the CRISPR/Cas9 method can be exploited as a powerful tool for livestock improvement by targeting multiple genes that are in charge of economically significant traits simultaneously.

Keywords: Cas9(Cas9), Sheep(绵羊), MSTN(MSTN), BCO2(BCO2), ASIP(ASIP)

Background

Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been used to modify many cell lines and organisms in the past. The recent CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) technology development provides an efficient tool for genome modifying of targeting loci. This system has an advantage of modifying multiple loci at the same time. Sheep with specific gene modifications may contribute to breeding. The results demonstrate the first detailed evidence of large animal modification in sheep.

Materials and Reagents

  1. Pipette tips
  2. Centrifuge tubes
  3. 6-well culture plate
  4. HiBindTM DNA Mini Column
  5. Sheep (Healthy ewes [3-5 years old] were selected about 160)
  6. PGL3-U6-gRNA vector (Addgene, catalog number: 51133 )
  7. pUC57-T7-gRNA vector (Addgene, catalog number: 51132 )
  8. E. coli DH5α competent cells
  9. Cas9 mRNA in vitro transcription vector (Addgene, catalog number: 44758 )
  10. T vector (Takara Bio, catalog number: D103A )
  11. Bsal enzyme(New England Biolabs, catalog number: R3535 )
  12. DNA Gel Extraction Kit (Corning, catalog number: D205-04 )
  13. T4 ligase (New England Biolabs)
  14. Ampicillin
  15. EndoFree Plasmid Maxi Kit (QIAGEN, catalog number: 12362 )
  16. Primers
    1. M13R (-47) primer
      5’-CGCCAGGGTTTTCCCAGTCACGAC-3’
    2. Primers for amplifying the selected potential off-target loci, designed by Primer5 software.


  17. Ethanol
  18. Ice acetic acid (Guangzhou Chemical Reagent Factory, catalog number: CB39-GR-0.5L )
  19. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM)
  20. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM)
  21. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM)
  22. Lipofectamine3000 reagent (Thermo Fisher Scientific, InvitrogenTM)
  23. Opti-MEM medium (Thermo Fisher Scientific, GibcoTM)
  24. Blasticidine S hydrochloride
  25. Universal Genomic DNA Kit (CWBIO, catalog number: CW2298M )
  26. KOD-Plus-Neo
  27. PCR cleanup kit (AppliChem, catalog number: A7089,1000 )
  28. T7EI
  29. Agarose
  30. TCM199 medium (Thermo Fisher Scientific, GibcoTM)
  31. Quinn’s Advantage Cleavage Medium and Blastocyst Medium (Sage Biopharma)
  32. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific)
  33. REPLI-g Single Cell Kit (QIAGEN, catalog number: 150343 )
  34. D2000 DNA marker
  35. Tris
  36. Ethylenediaminetetraacetate acid (EDTA)
  37. Agar powder
  38. Tryptone
  39. Yeast extract
  40. Sodium chloride (NaCl)
  41. 50x TAE (see Recipes)
  42. LB medium (see Recipes)

Equipment

  1. Pipettes
  2. 37 °C water bath
  3. Thermal cycler (Bio-Rad thermocycler)
  4. DNA electrophoresis apparatus
  5. Microcentrifuge
  6. Eppendorf FemtoJect system
  7. Olympus micromanipulation system ON3 (Olympus, model: ON3 SERIES )
  8. Microinjection device
  9. Biopsy forceps (Olympus, model: FB-11K-1 )

Software

  1. Primer5 software

Procedure

  1. sgRNA design
    1. Select specific target sites in sheep genome and then design sgRNA-oligo sequence.
    2. After design of sgRNA-oligo sequence, appropriate adaptor sequences should be added for cloning. The upstream primer of both pUC57-T7-gRNA vector and PGL3-U6-gRNA should include 5’-TAGG-3’ adaptor sequence, while the downstream primer for both should contain an adaptor with the sequence of 5’-AAAC-3’.

  2. Construct sgRNA-Cas9 plasmid
    1. Digest the PGL3-U6-gRNA vector and pUC57-T7-gRNA vector by BsaI.


    2. Incubate at 37 °C for 3-4 h.
    3. Purify the digested vector using DNA Gel Extraction Kit.
    4. Double-strand sequences and prepare double-strand oligo DNA reaction system:
      MSTN:
      sgRNA1:
      5’ CCAACTGTGGATATATCTGAGAC 3’


      5’ GTCTCAGATATATCCACAGTTGG 3’

      sgRNA2:
      5’ GGATTTTGAAGCTTTTGGATGG 3’


      5’ CCATCCAAAAGCTTCAAAATCC 3’
      ASIP:
      sgRNA1:
      5’ CCAGAGATGAAAGGAACCTGAAG 3’


      5’ CTTCAGGTTCCTTTCATCTCTGG 3’

      sgRNA2:
      5’ CAATTCTTCCATGAACCTGTTGG 3’


      5’ CCAACAGGTTCATGGAAGAATTG 3’
      BCO2:
      sgRNA1:
      5’ CCATGCATTGCACCGCTTCTAAC 3’


      5’ GTTAGAAGCGGTGCAATGCATGG 3’

       sgRNA2:
      5’ GGTCGTCTCAGCTCGAGTCCAGG 3’


      5’ CCTGGACTCGAGCTGAGACGACC 3’



    5. Perform PCR using the following cycling conditions:


    6. Ligate the diluted double-strand oligo DNA with the PGL3-U6-gRNA vector digested by BsaI:


    7. Incubate at 16 °C overnight.
    8. Transform 1 µl of ligation product into 50 µl E. coli DH5α competent cells.
    9. Pick up one colony and inoculate in a 15 ml tube containing 5 ml LB medium with 50 µg/ml ampicillin (4 colonies for each transformation). Incubate overnight in a 37 °C shaker.
    10. Extract plasmids from transformed DH5α cells using Endo-Free Plasmid Mini Kit following the manufacturer’s instructions (see Supplement 1).
    11. Confirm the positive clones containing PGL3-U6-gRNA and pUC57-T7-gRNA by Sanger sequencing using M13R (-47) primer.

  3. Cas9/sgRNA efficacy test in sheep fibroblast
    1. Sheep fibroblasts were obtained from a 40-day-old fetus and then transported to the laboratory.
    2. The fibroblasts are cultured for five passages in DMEM medium (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco) until 80-90% confluence.
    3. Cell transfection.
      1. The transfection procedure is performed using Lipofectamine3000 reagent (Invitrogen) according to the specification (Table 1).

        Table 1. The specification about transfection using Lipofectamine3000 Reagent (Invitrogen)


      2. Fibroblasts are separately transfected as followed types:


    4. Dilute by adding 10 mg/ml of blasticidin S hydrochloride into the medium (1:1,000 dilution) 24 h after transfection.
    5. Then incubate for 24 h at 37 °C.
    6. Genomic DNA is extracted from fibroblasts at 72 h after transfection using a Universal Genomic DNA Kit.
    7. Amplify targeted fragments using KOD polymerase, then purify with a PCR cleanup kit.
      1. Primers used for amplifying Cas9/sgRNA targeted MSTN, ASIP and BCO2 fragment.


      2. Prepare the reaction mixtures as follows:


      3. Perform PCR using the following conditions [annealing temperature (T) and extension time (L)].


    8. The purified PCR products are denatured and re-annealed in NEBuffer 2 (NEB) using a Bio-Rad thermocycler.
    9. The PCR products are digested with T7EI for 30 min at 37 °C and separated by 2.5% agarose gel electrophoresis.
    10. PCR products of mutations detected by T7EI cleavage assay are sub-cloned into T vector.
    11. For each sample, the colonies are randomly picked and sequenced using an M13F (-47) to confirm the positive colony.

  4. Off-target detection in sheep fibroblasts
    1. Predict the potential off-target sites using SeqMap.
    2. Extract sheep fibroblasts DNA for PCR amplification using specific primers (see Materials and Reagents).
      1. Mix the reaction components according to the follows:


      2. Perform PCR using the following conditions [annealing temperature (T) and extension time (L)].


    3. The PCR products are then subjected to a T7EI cleavage assay as described above and results below (Figure 1).


      Figure 1. Detection of the sgRNA:Cas9-mediated off-target cleavage of MSTN, ASIP, and BCO2 by using the T7EI cleavage assay. The PCR products of the potential off-target sites in MSTN, ASIP, and BCO2 sgRNA:Cas9 loci from sheep fibroblasts (A and B). 17 predicted off-target sites that were most homologous to MSTN, ASIP, and BCO2 sgRNAs were named OT1 to OT17. C and D. Detection of the sgRNA:Cas9-mediated off-target cleavage of MSTN, ASIP, and BCO2 by using the T7EI cleavage assay. More messages see Wang et al. (2016).

  5. Zygote collection
    Note: Healthy ewes (3-5 years old) were selected to be desired donors for zygote collection which include regular estrus cycles.
    1. Zygote collection and treatment of donors are conducted as earlier described in goats (Wang et al., 2015).
    2. Collect one cell stage zygotes (around 10-16 h after the last insemination) using a surgical way and immediately transfer the collected zygotes into a TCM199 medium.

  6. Efficient detection of sheep zygotes
    1. Microinject the sgRNA & Cas9 mRNA into sheep zygotes using an Olympus micromanipulation system ON3 in a manipulation medium TCM199 on the heated platform.
      Note: Mix Cas9 mRNA (20 ng/µl) and sgRNAs (5 ng/µl for each sgRNA) targeting MSTN, ASIP and BCO2.
    2. Inject the sgRNA & Cas9 mRNA into the cytoplasm of fertilized oocytes using an Eppendorf FemtoJect system with the following set-up.


    3. The embryos are cultured for 168 h at 38.5 °C in Quinn’s Advantage Cleavage Medium and Blastocyst Medium (Sage Biopharma).
    4. Blastocyst embryos are used for efficient detection preserved in PBS.
    5. Genomes of embryos are amplified using REPLI-g Single Cell Kit.
    6. Targeted fragments are amplified as described above including primers, reactions and procedures.
    7. Purification of PCR products, T7EI cleavage assay, and Sanger sequencing are performed according to Wang et al. (2015).

  7. Production of gene-modified sheep
    Note: Gene-modified sheep and control animals were fed the same diet, and managed at the same conditions throughout the experiment.
    1. Select the surrogate ewes with their natural estrus cycles as gist.
    2. Zygote collection and treatment of donors are conducted as described above.
    3. The zygotes are cultured for 24 h at 37 °C using Quinn’s Advantage Cleavage Medium (Sage Biopharma).
    4. Then culture the zygotes in Quinn’s Advantage Blastocyst Medium (Sage Biopharma) at 37 °C, 5% CO2 and saturated humidity.
    5. About 3-4 divisive embryos are transplanted into the ampullary-isthmic junction of the oviduct of the recipient ewes.
    6. Pregnancy normally takes about 150 days in Tan sheep, and is determined based on the observation of estrus behaviors in every ovulation cycle.

  8. Efficiency detection in Genetic-modified sheep
    1. Jugular venous blood samples and tissues are collected from randomly selected genetic-modified sheep (n = 10) and randomly selected control animals (n = 10) at 240 days of age for blood and tissues chemistry analyses.
    2. Genomic DNA is extracted from blood and tissues using corresponding assay from the University Genomic DNA Kit.
    3. Targeted fragments are amplified as described above in off-target detection in sheep fibroblast (including primers, reactions and procedures) (Procedure D).
    4. T7EI cleavage assay and sequencing are performed as previously described (step C9).
    5. The results may confirm the mutation in gene-modified sheep. (Figure 2)


      Figure 2. Evaluation of sgRNA:Cas9-mediated genetic modifications in lambs. A. PCR products of the targeted region of the MSTN, ASIP, and BCO2 genes of founder sheep co-microinjected with a mixture of Cas9 mRNA and sgRNAs. D2000 DNA marker was used as a marker reference. B. Detection of sgRNA:Cas9-mediated on-target cleavage of the MSTN, ASIP and BCO2 genes by using a T7EI cleavage assay. All PCR products from (A) were subjected to the T7EI cleavage assay. C. Sequencing results of the modified MSTN, ASIP and BCO2 loci that were detected in the founder animals. Target sequences complementary to sgRNAs of targeted genes are in red text, while the PAM sequences are marked in green. The mutations are marked in blue, dashlines indicates deletions, and lowercase indicates insertions or replacements. Insertions (+), deletions (-), mutations (m) is shown to the right of each allele. The genotypes are shown to the right with the rates of total clones for TA-sequencing. For more information see Wang et al. (2016).

  9. Germline transmission detection
    1. Germ cells from the testis of the three founders (#28, #33, and #37) were obtained by using biopsy forceps.
    2. The genomes of germ cells are amplified using REPLI-g Single Cell Kit.
    3. PCR products with mutations are detected using the T7EI cleavage assay, followed by Sanger sequencing.

Recipes

  1. 50x TAE
    Dissolve 242 g of Tris, 37.2 g of EDTA in 700 ml dH2O
    Note: Heat solution to 60 °C to assist in dissolution.
    Add 57.1 ml of ice acetic acid
    Add dH2O to 1,000 ml
    Then 200 ml solution was diluted to 1x TAE working solution by adding 10 L dH2O
  2. LB medium
    Dissolve 2 g agar powder, 2 g tryptone, 1 g yeast extract, 1 g NaCl in 200 ml dH2O

Acknowledgments

This work was supported by National Natural Science Foundation of China (31372279, 31402038, and 31171377), the Major Projects for New Varieties of Genetically Modified Organisms of China (2014ZX08008-002), as well as by China Agriculture Research System (CARS-40-13).

References

  1. Wang, X., Niu, Y., Zhou, J., Yu, H., Kou, Q., Lei, A., Zhao, X., Yan, H., Cai, B., Shen, Q., Zhou, S., Zhu, H., Zhou, G., Niu, W., Hua, J., Jiang, Y., Huang, X., Ma, B. and Chen, Y. (2016). Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep. Sci Rep 6: 32271.
  2. Wang, X., Yu, H., Lei, A., Zhou, J., Zeng, W., Zhu, H., Dong, Z., Niu, Y., Shi, B., Cai, B., Liu, J., Huang, S., Yan, H., Zhao, X., Zhou, G., He, X., Chen, X., Yang, Y., Jiang, Y., Shi, L., Tian, X., Wang, Y., Ma, B., Huang, X., Qu, L. and Chen, Y. (2015). Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system. Sci Rep 5: 13878.

简介

绵羊是研究生物医学研究发展和疾病的主要大动物模型。 我们成功地利用CRISPR / Cas9系统修改了绵羊中的多个基因。 在这里,我们提供一个细胞阶段胚胎手术的详细协议,通过共同注射针对三个基因的Cas9 mRNA和RNA指南(MSTN ,ASIP 和 BCO2 )来创建遗传修饰的绵羊。 程序描述了sgRNA设计,gRNA-Cas9质粒的构建,在成纤维细胞,胚胎和绵羊中的有效检测以及一些操纵技术。 我们的研究结果表明,CRISPR / Cas9方法可以作为强大的家畜改良工具,同时针对多个负责经济重要性状的基因。
【背景】锌指核酸酶(ZFN)和转录激活物样效应核酸酶(TALENs)已被用于修改过去许多细胞系和生物体。 最近的CRISPR / Cas(集群定期间隔短回文重复/ CRISPR相关)技术开发为靶基因座的基因组修饰提供了有效的工具。 该系统具有同时修改多个基因座的优点。 具有特定基因修饰的绵羊可能有助于育种。 结果表明羊的大动物修饰的第一个详细的证据。

关键字:Cas9, 绵羊, MSTN, BCO2, ASIP

材料和试剂

  1. 移液器提示
  2. 离心管
  3. 6孔培养板
  4. HiBindTM DNA迷你柱
  5. 羊(健康的母羊[3-5岁]被选中约160)
  6. PGL3-U6-gRNA载体(Addgene,目录号:51133)
  7. pUC57-T7-gRNA载体(Addgene,目录号:51132)
  8. 电子。大肠杆菌DH5α感受态细胞
  9. Cas9 mRNA体外转录载体(Addgene,目录号:44758)
  10. T载体(Takara Bio,目录号:D103A)
  11. Bsa l酶(New England Biolabs,目录号:R3535)
  12. DNA凝胶提取试剂盒(Corning,目录号:D205-04)
  13. T4连接酶(New England Biolabs)
  14. 氨苄青霉素
  15. EndoFree Plasmid Maxi Kit(QIAGEN,目录号:12362)
  16. 引物
    1. M13R(-47)底漆
      5'-CGCCAGGGTTTTCCCAGTCACGAC-3'
    2. 用于放大选定的潜在离靶位点的引物,由Primer5软件设计。


  17. 乙醇
  18. 冰醋酸(广州化学试剂厂,目录号:CB39-GR-0.5L)
  19. Dulbecco改性Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM
  20. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM
  21. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM
  22. Lipofectamine3000试剂(Thermo Fisher Scientific,Invitrogen TM
  23. Opti-MEM培养基(Thermo Fisher Scientific,Gibco TM
  24. 杀虫剂S盐酸盐
  25. 通用基因组DNA试剂盒(CWBIO,目录号:CW2298M)
  26. KOD-Plus-Neo
  27. PCR清除试剂盒(AppliChem,目录号:A7089,1000)
  28. T7EI
  29. 琼脂糖
  30. TCM199培养基(Thermo Fisher Scientific,Gibco TM
  31. Quinn的优势切割介质和囊胚培养基(Sage Biopharma)
  32. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific)
  33. REPLI-g单电池套件(QIAGEN,目录号:150343)
  34. D2000 DNA标记
  35. Tris
  36. 乙二胺四乙酸(EDTA)
  37. 琼脂粉末
  38. 胰蛋白胨
  39. 酵母提取物
  40. 氯化钠(NaCl)
  41. 50x TAE(见配方)
  42. LB培养基(见食谱)

设备

  1. 移液器
  2. 37°C水浴
  3. 热循环仪(Bio-Rad thermocycler)
  4. DNA电泳仪
  5. 微量离心机
  6. Eppendorf FemtoJect系统
  7. 奥林巴斯微操作系统ON3(奥林巴斯,型号:ON3系列)
  8. 显微注射装置
  9. 活检钳(Olympus,型号:FB-11K-1)

软件

  1. Primer5软件

程序

  1. sgRNA设计
    1. 选择羊基因组中的特定目标位点,然后设计sgRNA-寡核苷酸序列
    2. 在设计sgRNA-oligo序列后,应加入适当的衔接子序列进行克隆。 pUC57-T7-gRNA载体和PGL3-U6-gRNA的上游引物应包括5'-TAGG-3'衔接子序列,而两者的下游引物应包含具有5'-AAAC-3' 。

  2. 构建sgRNA-Cas9质粒
    1. 通过Bsa I来消化PGL3-U6-gRNA载体和pUC57-T7-gRNA载体。


    2. 在37°C孵育3-4小时。
    3. 使用DNA凝胶提取试剂盒纯化消化的载体
    4. 双链序列并制备双链寡DNA反应体系:
      MSTN:
      sgRNA1:
      5’ CCAACTGTGGATATATCTGAGAC 3’


      5’ GTCTCAGATATATCCACAGTTGG 3’

      sgRNA2:
      5’ GGATTTTGAAGCTTTTGGATGG 3’


      5’ CCATCCAAAAGCTTCAAAATCC 3’
      ASIP:
      sgRNA1:
      5’ CCAGAGATGAAAGGAACCTGAAG 3’


      5’ CTTCAGGTTCCTTTCATCTCTGG 3’

      sgRNA2:
      5’ CAATTCTTCCATGAACCTGTTGG 3’


      5’ CCAACAGGTTCATGGAAGAATTG 3’
      BCO2:
      sgRNA1:
      5’ CCATGCATTGCACCGCTTCTAAC 3’


      5’ GTTAGAAGCGGTGCAATGCATGG 3’

       sgRNA2:
      5’ GGTCGTCTCAGCTCGAGTCCAGG 3’


      5’ CCTGGACTCGAGCTGAGACGACC 3’



    5. 使用以下循环条件进行PCR:


    6. 用通过Bsa I消化的PGL3-U6-gRNA载体来连接稀释的双链寡核苷酸:


    7. 在16℃下孵育过夜。
    8. 将1μl连接产物转化成50μlE.大肠杆菌DH5α感受态细胞。
    9. 取一个菌落并接种在含有50μg/ ml氨苄青霉素(每个转化4个菌落)的5ml LB培养基的15ml管中。在37°C振荡器中孵育过夜。
    10. 使用Endo-Free Plasmid Mini Kit从转化的DH5α细胞中提取质粒,遵循制造商的说明(参见补充1 )。
    11. 使用M13R(-47)引物,通过Sanger测序确认含有PGL3-U6-gRNA和pUC57-T7-gRNA的阳性克隆。

  3. 绵羊成纤维细胞Cas9 / sgRNA功效试验
    1. 绵羊成纤维细胞从40天龄的胎儿获得,然后运送到实验室。
    2. 在补充有10%FBS(Gibco)和1%青霉素 - 链霉素(Gibco)的DMEM培养基(Gibco)中将成纤维细胞培养5代,直到80-90%汇合。
    3. 细胞转染。
      1. 根据说明书(表1),使用Lipofectamine3000试剂(Invitrogen)进行转染过程。

        表1.使用Lipofectamine3000试剂(Invitrogen)进行转染的规范


      2. 成纤维细胞分别转染为以下类型:


    4. 转染24小时后,向培养基中加入10mg / ml杀稻瘟素S盐酸盐稀释至稀释液(1:1,000)。
    5. 然后在37°C孵育24 h
    6. 使用通用基因组DNA试剂盒转染后72 h,从成纤维细胞中提取基因组DNA
    7. 使用KOD聚合酶扩增靶向片段,然后用PCR清除试剂盒进行纯化。
      1. 用于扩增靶向MSTN ,ASIP 和/或BCO2 片段的Cas9 / sgRNA的引物。


      2. 如下制备反应混合物:


      3. 使用以下条件进行PCR [退火温度(T)和延长时间(L)]

    8. 纯化的PCR产物在NEBuffer 2(NEB)中使用Bio-Rad热循环仪进行变性和再退火。
    9. PCR产物在37℃下用T7EI消化30分钟,并通过2.5%琼脂糖凝胶电泳分离。
    10. 通过T7EI切割测定检测到的突变的PCR产物被亚克隆到T载体中
    11. 对于每个样品,随机挑选菌落并使用M13F(-47)测序以确认阳性菌落。

  4. 羊成纤维细胞中的脱靶检测
    1. 使用SeqMap预测潜在的目标外站点。
    2. 使用特异性引物提取绵羊成纤维细胞DNA进行PCR扩增(参见材料和试剂)。
      1. 按照下列步骤混合反应组分:


      2. 使用以下条件进行PCR [退火温度(T)和延长时间(L)]

    3. 然后将PCR产物进行如上所述的T7EI切割测定,结果如下(图1)

      图1. sgRNA的检测:Cas9介导的off靶标切割 MSTN 通过使用T7EI切割测定法,强> ASIP BCO2 来自绵羊成纤维细胞(A和B)的MSTN,ASIP 和bCO2 sgRNA:Cas9位点的潜在off靶位点的PCR产物。与MSTN ,ASIP 和 BCO2 sgRNAs最相似的off-靶位点的预测命名为OT1至OT17。 C和D.通过使用T7EI切割测定法检测sgRNA:Cas9介导的MSTN ,ASIP 和/或BCO2靶向切割。更多消息请参阅 Wang 等。(2016)

  5. 合辑集合
    注意:选择健康的母羊(3-5岁)作为合适的受精卵供体,包括定期发情周期。
    1. 献血者的合子收集和治疗如前所述在山羊中进行(Wang等人,2015)。
    2. 使用外科手术收集一个细胞阶段受精卵(最后一次授精约10-16小时),并立即将收集的合子转移到TCM199培养基中。

  6. 有效检测羊受精卵
    1. 显微注射sgRNA&在加热平台上的操作介质TCM199中使用Olympus显微操作系统ON3将Cas9 mRNA转入羊合子。
      注意:将针对MSTN,ASIP和BCO2的Cas9 mRNA(20ng /μl)和sgRNA(针对每个sgRNA的5ng /μl)混合。/ / em>
    2. 注射sgRNA和Cas9 mRNA使用Eppendorf FemtoJect系统进入受精卵细胞的细胞质,并进行以下设置:


    3. 在Quinn的优势切割培养基和胚泡培养基(Sage Biopharma)中,将胚胎在38.5℃培养168小时。
    4. 胚胎胚胎用于在PBS中保存的有效检测。
    5. 使用REPLI-g单细胞试剂盒扩增胚胎基因组。
    6. 如上所述扩增靶向片段,包括引物,反应和程序
    7. PCR产物的纯化,T7EI切割测定和Sanger测序根据Wang等人(2015)进行。

  7. 基因修饰羊的生产
    注意:基因修饰的羊和对照动物喂食相同的饮食,并在整个实验中在相同的条件下进行管理。
    1. 选择以自然发情循环为代表的代孕母羊。
    2. 如上所述进行给药者的合子收集和治疗。
    3. 使用Quinn的优势切割培养基(Sage Biopharma),将受精卵在37℃下培养24小时。
    4. 然后在Quinn的优势囊胚培养基(Sage Biopharma)中培养受精卵,37℃,5%CO 2和饱和湿度。
    5. 大约3-4个分裂的胚胎被移植到接受母羊的输卵管的壶腹 - 交界处。
    6. 妊娠期Tan羊通常需要约150天,根据每个排卵周期的发情行为情况确定。

  8. 遗传修饰羊的效率检测
    1. 从240日龄随机选择的遗传修饰的绵羊(n = 10)和随机选择的对照动物(n = 10)收集颈静脉血样和组织进行血液和组织化学分析。
    2. 使用大学基因组DNA试剂盒的相应测定法从血液和组织中提取基因组DNA
    3. 如上所述,靶向片段在绵羊成纤维细胞(包括引物,反应和程序)的脱靶检测中扩增(方法D)。
    4. 如前所述进行T7EI切割测定和测序(步骤C9)
    5. 结果可以证实基因修饰的羊的突变。 (图2)


      图2. sgRNA的评估:Cas9介导的羔羊遗传修饰。 :一种。用Cas9 mRNA和sgRNA的混合物共同显微注射的创始人羊的MSTN ,,ASIP 和 BCO2 基因的目标区域的PCR产物。 D2000 DNA标记用作标记参考。 B.通过使用T7EI切割测定法检测sgRNA:Cas9介导的MSTN,ASIP和/或BCO2基因的目标上切割。将来自(A)的所有PCR产物进行T7EI切割测定。 C.在创始人动物中检测到的经修改的MSTN , ASIP 和 BCO2 基因座的测序结果。与目标基因的sgRNA互补的靶序列是红色文本,而PAM序列标记为绿色。突变标记为蓝色,短划线表示缺失,小写表示插入或替换。插入(+),缺失( - ),突变(m)显示在每个等位基因的右侧。基因型显示为右侧与TA测序的总克隆的比率。有关更多信息,请参阅 Wang 等。(2016)

  9. 种系传播检测
    1. 通过使用活检钳获得三名创始人(#28,#33和#37)睾丸的生殖细胞。
    2. 使用REPLI-g单细胞试剂盒扩增生殖细胞的基因组。
    3. 使用T7EI切割测定法检测具有突变的PCR产物,随后进行Sanger测序。

食谱

  1. 50倍TAE
    将242g Tris,37.2g EDTA溶解在700ml dH 2 O中,/ / 注意:将溶液加热至60°C以帮助溶解。
    加入57.1毫升冰醋酸 将dH 2 O添加到1,000 ml
    然后,将200ml溶液通过加入10μLdH 2 O→/
  2. LB培养基
    将2g琼脂粉末,2g胰蛋白胨,1g酵母提取物,1g NaCl的200ml dH 2 O

致谢

这项工作得到了中国国家自然科学基金(31372279,31402038和31171377),中国转基因生物新品种重大项目(2014ZX08008-002)以及中国农业科研体系(CARS-40)的支持。 -13)。

参考

  1. Wang,X.,Niu,Y.,Zhou,J.,Yu,H.,Kou,Q.,Lei,A.,Zhao,X.,Yan,H.,Cai,B.,Shen, Zhou,S.,Zhu,H.,Zhou,G.,Niu,W.,Hua,J.,Jiang,Y.,Huang,X.,Ma,B. and Chen,Y。(2016) 通过CRISPR / Cas9进行多重基因编辑显示出所需的肌肉肥大,无需检测 - 羊的目标效应。 Sci Rep 6 :32271.
  2. Wang,X.,Yu,H.,Lei,A.,Zhou,J.,Zeng,W.,Zhu,H.,Dong,Z.,Niu,Y.,Shi,B.,Cai, Liu,J.,Huang,S.,Yan,H.,Zhao,X.,Zhou,G.,He,X.,Chen,X.,Yang,Y.,Jiang,Y.,Shi, Tian,X.,Wang,Y.,Ma,B.,Huang,X.,Qu,L. and Chen,Y。(2015)。通过合格注射CRISPR生成靶向MSTN 和 FGF5 的基因修饰山羊/ Cas9系统。 Sci Rep 5:13878.
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Niu, Y., Ding, Y., Wang, X. and Chen, Y. (2017). Multiplex Gene Editing via CRISPR/Cas9 System in Sheep. Bio-protocol 7(13): e2385. DOI: 10.21769/BioProtoc.2385.
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