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Genotyping-free Selection of Double Allelic Gene Edited Medaka Using Two Different Fluorescent Proteins
使用两种不同的荧光蛋白进行双等位基因编辑青鳉的无基因分型选择   

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参见作者原研究论文

本实验方案简略版
Zoological Letters
Jul 2017

Abstract

This protocol describes a simple genotyping using two different colors of fluorescent protein genes inserted at the target locus. This method makes it possible to determine the genotype of each individual simply by observing the fluorescence later than F1 generation.

Keywords: Medaka (青鳉), Genome editing (基因组编辑), Gene knock-in (基因敲入), CRISPR/Cas (CRISPR/Cas), Fluorescent reporter gene (荧光报告基因)

Background

Because the conventional genotyping of genome edited animals largely depends on PCR-based genotyping, it is necessary to extract genomic DNA. In medaka, tail fin is an accessible and regenerable tissue, so that it is frequently used for genotyping to keep fish alive. To cut tail fin, fish must be fed for a few weeks to grow big enough (ca. 1 cm in body length). Feeding and taking care of many fishes are time-consuming and laborious. Therefore, reducing the number of fish to be fed in the early stage of breeding, such as embryos and early larvae, is desired to reduce the tasks. However, it is hard to take tail fin from embryos and early larvae alive because they are too small and fragile. To overcome this issue, a simple and non-invasive method for genotyping is essential. Therefore, we developed a new method that can non-invasively determine the genotype of each individual at embryonic stage simply by observing the fluorescence. We demonstrated the benefit of this method in a gene knock-in experiment targeting the growth associated protein 43 (gap43) gene expressed in the central nervous system (CNS) at 4 days post fertilization (dpf) (Murakami et al., 2017). This gene is suitable for the gene knock-in experiment, because its spatio-temporal expression pattern has been already revealed by RT-PCR and in situ hybridization in a previous study (Fujimori et al., 2008). If the expression pattern of a targeted gene in wild-type is unknown, it needs to be analyzed by RT-PCR, or any other technique, in order to confirm the correspondence between it and that of reporter gene in gene knock-in strains.

Materials and Reagents

  1. Plasmid pDR274 (Addgene, catalog number: 42250 )
  2. pBaitD-gap43-linker-EGFP (RIKEN DNA BANK, catalog number: RDB15409 )
  3. Ampliscribe T7-Flash Transcription Kit (Epicentre, catalog number: ASF3257 )
  4. RNeasy Plus Mini Kit (QIAGEN, catalog number: 74134 )
  5. PCR-Kit; KOD-Plus-Neo (TOYOBO, catalog number: KOD-401 )
  6. Sodium hydroxide (NaOH) (NACALAI TESQUE, catalog number: 31511-05 )
  7. Ethylenediaminetetraacetate acid (EDTA) (NACALAI TESQUE, catalog number: 14347-21 )
  8. Tris-HCl (pH 8.0) (NACALAI TESQUE, catalog number: 35435-11 )
  9. Alkaline lysis buffer (see Recipes)
  10. Neutralization buffer (see Recipes)

Equipment

  1. Forceps (DUMONT, model: 91-3869 or equivalents)
  2. PCR thermal cycler (NIPPON Genetics, catalog number: TC-96GHbC )
  3. Incubator (NKsystem, catalog number: LH-60FL3-DT )
  4. Fluorescent microscope (Leica, catalog number: Leica MZ8 )

Procedure

  1. Search target sequences for gRNAs using a gRNA-scoring algorithm (CRISPRscan; Moreno-Mateos et al., 2015), and select the one that has less off-target sites in medaka genome.
  2. Obtain oligonucleotides for the selected gRNA from a supplier of choice.
  3. Anneal the oligonucleotides and insert the annealed fragment into gRNA expression plasmid pDR274 (Addgene Plasmid #42250).
  4. Transcribe from the DraI-digested plasmid using the Ampliscribe T7-Flash Transcription Kit, and purify the transcript with the RNeasy Plus Mini Kit to eliminate the template DNA without DNase treatment.
  5. PCR-amplify the upstream and downstream regions (homology arms: ca. 500 bp) of the genomic target site using the region-specific primers with restriction enzyme sites, which help to insert into the donor plasmid described in Step 6.
  6. Ligate both the homology arms, the insert fragment containing a linker, a reporter gene, and a polyA signal into the backbone plasmid containing the BaitD sequence that helps to induce gene knock-in events with high efficiency in medaka (Figure 1A).
    Note: The donor plasmid containing BaitD can be obtained from RIKEN DNA BANK (pBaitD-gap43-linker-EGFP #15409). The sequence of the donor plasmid is shown as Supplemental file 1.


    Figure 1. Schematic illustration of a gene knock-in experiment. A. Schematic design of the donor plasmid. The left (blue in (A)) and right (green in (A)) homology arms (ca. 500 bp) are amplified respectively using the region-specific primers with restriction enzyme sites (XhoI/BamHI or EcoRI/SpeI). Linker sequence stabilizes to express the reporter gene (GFP or RFP) as a fusion protein with the target gene product. Bait is a sequence to be cleaved for linearization of the donor plasmid, and enhances gene knock-in events in medaka. Thunder marks show the cleavage sites on the genome and the donor plasmid by Cas9 and gRNA1 or gRNA2, respectively. B. Schematic illustration showing precise integration of the insert gene into the genomic target site. The reporter gene (GFP or RFP) is integrated into exon and in-frame. Blue and green triangles show the junctions between the genomic target site and the fragment of donor plasmid. Primer pairs labeled (1)/(2) or (3)/(4) can be used for amplifying the upstream or downstream junction region. To confirm the precise integration into the genomic target site, primer (1) and (4) are designed on the outside of the junctions.

  7. Inject the following solution into embryos; 100 ng/µl of Cas9 RNA, 50 ng/µl of sgRNAs for cleaving the genomic target site and the donor plasmid, and 2.5 ng/µl of each donor plasmid.
    Note: Please refer to ‘Medaka-microinjection’ for the details of microinjection protocol.
  8. Raise the embryos expressing each reporter gene to adult for about 2 months.
  9. Mate with wild type counterparts. (First mating)
  10. Extract genomic DNA from the resultant F1 embryos expressing each reporter gene, according to the following protocol:
    1. Put embryos in 25 µl of alkaline lysis buffer (see Recipes).
    2. Incubate at 95 °C for 15 min after breaking the egg envelope with forceps.
    3. Neutralize with 25 µl of neutralized buffer (see Recipes).
    Note: DNA extraction is only necessary for F1 generation, because it is needed to investigate by the sequence analysis whether the insert gene is correctly integrated into the genomic target site.
  11. PCR-amplify the junction regions of the target site on the host genome and the introduced gene using F1 genomic DNA (Figure 1B).
    Note: The PCR condition is performed as described in the protocol of KOD-Plus-Neo (Toyobo).
  12. Perform the sequence analysis of the resultant PCR amplicons, to confirm the precise integration into the genomic target site.
  13. After identifying F0 founders with the desired mutation, mate with wild type counterparts again. (Second mating)
    Note: First mating is for the identification of F0 founders harboring the insert gene in germ cells, while second mating is for the establishment of gene knock-in strains in F1 generation.
  14. Collect F1 embryos expressing each reporter gene, and raise to fish with a length > 1 cm.
  15. Cut off tail fin from each F1 fish, and perform the genome extraction and the sequence analysis described as above.
  16. After confirming the precise integration into the genomic target site in each F1 fish by the sequence analysis, mate F1 fish harboring different color reporter genes with each other.
  17. Observe the fluorescence color of the resultant F2 embryos, to specify the individuals integrated the insert gene on the target site.
    Note: The resultant F2 embryos are genotyped alive by fluorescence color: wild type without fluorescence, monoallelic mutants for integration with one fluorescence, and biallelic mutants for integration with both fluorescence.

Recipes

  1. Alkaline lysis buffer
    25 mM NaOH
    0.2 mM EDTA
  2. Neutralization buffer
    40 mM Tris-HCl (pH 8.0)

Acknowledgments

This protocol was adapted from our previous works (Murakami et al., 2017). The work was partially supported by a Grant-in-Aid for Scientific Research (A) 15H02540 (MK) and a Grant-in-Aid for JSPS fellows 13J01682 (SA). The funders had no role in the design of the study and collection, analysis, or interpretation of data or in the writing of the manuscript. The authors declare no conflicts of interest or competing interests with this manuscript.

References

  1. Fujimori, K. E., Kawasaki, T., Deguchi, T. and Yuba, S. (2008). Characterization of a nervous system-specific promoter for growth-associated protein 43 gene in Medaka (Oryzias latipes). Brain Res 1245: 1-15.
  2. Moreno-Mateos, M. A., Vejnar, C. E., Beaudoin, J. D., Fernandez, J. P., Mis, E. K., Khokha, M. K. and Giraldez, A. J. (2015). CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat Methods 12(10): 982-988.
  3. Murakami, Y., Ansai, S., Yonemura, A. and Kinoshita, M. (2017). An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes). Zoological Lett 3: 10.

简介

该协议描述了使用两种不同颜色的荧光蛋白基因插入目标基因座的简单基因分型。这种方法可以简单地通过观察比F1代更晚的荧光来确定每个个体的基因型。

【背景】由于基因组编辑动物的常规基因分型在很大程度上取决于基于PCR的基因分型,所以有必要提取基因组DNA。在青aka中,尾鳍是一种易于再生的组织,因此经常用于基因分型以保持鱼的活力。要切断尾鳍,必须喂鱼几周,使其长得足够大(体长约1厘米)。喂养和照顾许多鱼费时费力。因此,希望减少繁殖早期如胚和早期幼虫的鱼的数量,以减少任务。然而,由于胚胎和幼虫的尾鳍太小而脆弱,因此很难将它们活下来。为了克服这个问题,基因分型的一个简单和非侵入性的方法是必不可少的。因此,我们开发了一种新的方法,只需通过观察荧光就可以无创地确定每个胚胎期的基因型。我们在基因敲入实验中证实了该方法的益处,该实验针对在受精后4天(dpf)(Murakami)在中枢神经系统(CNS)中表达的生长相关蛋白43( gap43 et al 。,2017)。这个基因适用于基因敲入实验,因为在先前的研究中,已经通过RT-PCR和原位杂交揭示了其时空表达模式(Fujimori等, 。,2008)。如果野生型靶基因的表达模式是未知的,则需要通过RT-PCR或任何其他技术来分析,以证实其与基因敲入菌株中报道基因的对应性。

关键字:青鳉, 基因组编辑, 基因敲入, CRISPR/Cas, 荧光报告基因

材料和试剂

  1. 质粒pDR274(Addgene,目录号:42250)
  2. pBaitD-gap43-linker-EGFP(RIKEN DNA BANK,目录号:RDB15409)
  3. Ampliscribe T7-Flash转录试剂盒(Epicentre,目录号:ASF3257)
  4. RNeasy Plus迷你试剂盒(QIAGEN,产品目录号:74134)
  5. PCR-Kit的; KOD-Plus-Neo(TOYOBO,货号:KOD-401)
  6. 氢氧化钠(NaOH)(NACALAI TESQUE,目录号:31511-05)
  7. 乙二胺四乙酸(EDTA)(NACALAI TESQUE,目录号:14347-21)
  8. Tris-HCl(pH8.0)(NACALAI TESQUE,目录号:35435-11)
  9. 碱性裂解缓冲液(见食谱)
  10. 中和缓冲液(见食谱)

设备

  1. 镊子(DUMONT,型号:91-3869或等价物)
  2. PCR热循环仪(NIPPON Genetics,目录号:TC-96GHbC)
  3. 孵化器(NK系统,目录号:LH-60FL3-DT)
  4. 荧光显微镜(徕卡,目录号:徕卡MZ8)

程序

  1. 使用gRNA评分算法(CRISPRscan; Moreno-Mateos等人,2015)搜索gRNA的靶序列,并选择在青aka基因组中具有较少脱靶位点的序列。
  2. 从选择的供应商获得选定的gRNA寡核苷酸。
  3. 退火寡核苷酸并将退火的片段插入到gRNA表达质粒pDR274(Addgene Plasmid#42250)中。
  4. 使用Ampliscribe T7-Flash转录试剂盒从DraI I消化的质粒转录,并用RNeasy Plus Mini试剂盒纯化转录物以消除不用DNase处理的模板DNA。
  5. 使用具有限制酶位点的区域特异性引物PCR扩增基因组靶位点的上游和下游区域(同源臂:500bp),其有助于插入到在第6步。
  6. 将含有接头,报道基因和polyA信号的同源臂,插入片段连接到包含BaitD序列的骨架质粒中,有助于在青aka中高效诱导基因敲入事件(图1A)。
    注意:含有BaitD的供体质粒可以从RIKEN DNA BANK(pBaitD-gap43-linker-EGFP#15409)获得。供体质粒的序列显示为:补充文件1

    图1.基因敲入实验的示意图A.供体质粒的示意图设计。使用具有限制酶位点的区域特异性引物分别扩增左(蓝色(A))和右(绿色(A))同源臂(500 Xho I / Bam HI或 Eco RI / I)。接头序列稳定以表达报告基因( GFP 或 RFP )作为与目标基因产物的融合蛋白。诱饵是供体质粒线性化而被切割的序列,并且增强了青gene中的基因敲入事件。雷霆标记通过Cas9和gRNA1或gRNA2分别显示基因组和供体质粒上的切割位点。 B.显示插入基因精确整合到基因组靶位点的示意图。报告基因( GFP 或 RFP )被整合到外显子和框内。蓝色和绿色三角形显示基因组靶位点和供体质粒片段之间的连接点。标记为(1)/(2)或(3)/(4)的引物对可用于扩增上游或下游连接区。为了确认与基因组靶位点的精确整合,在接合处外侧设计了引物(1)和(4)。

  7. 注入以下解决方案到胚胎; 100ng /μl的Cas9 RNA,50ng /μl的用于切割基因组靶位点和供体质粒的sgRNA以及2.5ng /μl的每种供体质粒。
    注意:有关显微注射协议的详细信息,请参阅“ Medaka-microinjection ”。

  8. 提高表达每个记者基因的胚胎成人约2个月。
  9. 与野生型同行交配。 (第一次交配)
  10. 根据以下方案从表达每个报道基因的所得F1胚胎提取基因组DNA:
    1. 把胚胎放入25μl的碱裂解缓冲液(见食谱)。
    2. 用镊子打破蛋壳后,在95°C孵育15分钟。
    3. 用25μl中和缓冲液中和(见食谱)。
    注意:DNA提取仅对于F1代是必需的,因为需要通过序列分析来研究插入基因是否正确整合到基因组靶位点中。
  11. 使用F1基因组PCR扩增宿主基因组上目标位点的连接区和导入的基因(图1B)。 注意:PCR条件按照KOD-Plus-Neo(Toyobo)的方案进行。
  12. 执行所得PCR扩增子的序列分析,以确认精确整合到基因组目标位点。
  13. 在确定F0的创始人与所需的突变之后,再次与野生型同行交配。 (第二次交配)
    注:第一次交配是为了在生殖细胞中鉴定携带插入基因的F0创始者,而第二次交配是为了在F1代中建立基因敲入株。
  14. 收集表达每个报道基因的F1胚胎,并饲养长度> 1厘米。

  15. 切断每条F1鱼的尾鳍,进行如上所述的基因组提取和序列分析
  16. 在通过序列分析确认每个F1鱼的基因组目标位点的精确整合之后,将携带不同颜色记者基因的F1鱼彼此交配。
  17. 观察所得F2胚胎的荧光颜色,以指定将插入基因整合到目标位点上的个体。
    注:产生的F2胚胎通过荧光颜色进行活的基因分型:无荧光的野生型,与一种荧光整合的单等位基因突变体以及与两种荧光整合的双等位基因突变体。

食谱

  1. 碱性裂解液
    25mM NaOH
    0.2mM EDTA
  2. 中和缓冲液
    40mM Tris-HCl(pH8.0)

致谢

这个协议是从我们以前的作品(Murakami等人,2017年)改编的。这项工作部分得到了科学研究资助(A)15H02540(MK)和JSPS研究员13J01682(SA)的资助。资助者在设计研究和收集,分析或解释数据或撰写稿件方面没有任何作用。作者声明与本手稿没有利益冲突或利益冲突。

参考

  1. Fujimori,K.E.,Kawasaki,T.,Deguchi,T。和Yuba,S。(2008)。 青蒿中生长相关蛋白43基因神经系统特异性启动子的特征( Oryzias latipes )。 Brain Res 1245:1-15。
  2. Moreno-Mateos,M.A.,Vejnar,C.E.,Beaudoin,J.D.,Fernandez,J.P.,Mis,E.K。,Khokha,M.K。和Giraldez,A.J。(2015)。 CRISPRscan:为CRISPR-Cas9设计针对体内的高效sgRNAs 。 Nat Methods 12(10):982-988。
  3. Murakami,Y.,Ansai,S.,Yonemura,A.和Kinoshita,M.(2017)。 一种高效的青aka同源依赖靶向基因整合系统( Oryzias latipes )。 Zoological Lett 3:10。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Murakami, Y., Ansai, S., Yonemura, A. and Kinoshita, M. (2017). Genotyping-free Selection of Double Allelic Gene Edited Medaka Using Two Different Fluorescent Proteins. Bio-protocol 7(24): e2665. DOI: 10.21769/BioProtoc.2665.
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