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A Protocol for Production of Mutant Mice Using Chemically Synthesized crRNA/tracrRNA with Cas9 Nickase and FokI-dCas9
利用化学合成的crRNA/tracrRNA-Cas9 Nick酶及FokI-dCas9产生突变小鼠的实验方法   

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Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is the most widely used genome editing tool. A common CRISPR/Cas9 system consists of two components: a single-guide RNA (sgRNA) and Cas9. Both components are required for the introduction of a double-strand break (DSB) at a specific target sequence. One drawback of this system is that the production of sgRNA in the laboratory is laborious since it requires cloning of an sgRNA sequence, in vitro transcription reaction and sgRNA purification. An alternative to targeting Cas9 activity by sgRNA is to target it with two small RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Both of these small RNAs can be chemically synthesized which makes the production of these RNAs less difficult when compared to sgRNA. Another downside of the CRISPR/Cas9 systems is that off-target effects have been reported. However, modified forms of Cas9 have been developed to minimize off-target effects. For example, nickase-type Cas9 (nCas9) and FokI domain-fused catalytically-inactive Cas9 (FokI-dCas9; fCas9) induce DSBs only when two guide RNAs bind opposite strands within a defined distance. In this protocol, we describe our experimental system for the production of mutant mice using a CRISPR/Cas9 system that combines crRNA, tracrRNA, and modified forms of Cas9. This method not only facilitates the preparation of reagents for the genome editing system but it can also reduce the risk of off-target effects.

Keywords: CRISPR/Cas9(CRISPR / Cas9), crRNA/tracrRNA( crRNA / tracrRNA), nCas9( nCas9), fCas9( fCas9), Mutant mice(突变小鼠)

Background

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is an effective genome editing tool. In bacteria, CRISPR/Cas9 functions as an adaptive immune system. It consists of two small RNAs, CRISPR RNA (crRNA) and trans-activation crRNA (tracrRNA) and the Cas9 DNA nuclease, which digests targeted DNA (Jinek et al., 2013). Several groups have established the CRISPR/Cas9 system as a tool for introducing mutations in many cell types (Cong et al., 2013; Mali et al., 2013). When the Cas9 nuclease is targeted to genomic DNA, it cleaves DNA resulting in a lesion that is repaired by non-homologous end joining (NHEJ) or homologous DNA recombination. Since NHEJ can be an error-prone mechanism, mutations can be introduced into the genome when DNA is repaired by this mechanism. The CRISPR/Cas9 system can be used to edit the genomes of mice by microinjecting Cas9 and the single-guide RNA (sgRNA) into fertilized eggs. Although sgRNAs have been used extensively with success, the generation of the sgRNA is laborious because the sgRNA must be cloned from DNA oligomers and then transcribed in vitro. Systems that use crRNA and tracrRNA can eliminate much of the labor involved in preparing sgRNAs since crRNA and tracrRNA are small enough in length to be chemically synthesized. So instead of injecting Cas9 with sgRNA, mutant mice can be obtained by microinjecting Cas9 with both crRNA and tracrRNA (crRNA/tracrRNA). One drawback of using the CRISPR/Cas9, is guide RNA/Cas9 complex can generate off-target mutations, which are unintended mutations that occur at loci with similar sequences to the target sequence of the guide RNA. The risk of inducing off-target mutations can be reduced by incorporating the use of modified forms of Cas9, such as the nickase-type of Cas9 (nCas9) and the FokI domain-fused catalytically-inactive Cas9 (FokI-dCas9; fCas9). These Cas9 forms cleave target DNA only when two sgRNAs bind opposite strands with a limited distance between them (Ran et al., 2013; Hara et al., 2015). Recently, we successfully generated mutant mice by microinjecting modified Cas9s with chemically synthesized crRNA/tracrRNA into fertilized eggs (Terao et al., 2016). This protocol reduces time and labor for the preparation of targeting RNA and can reduce the risk of off-target effects.

Materials and Reagents

  1. 35 mm dish*
  2. 60 mm dish*
  3. Mouth pipettes*
  4. 0.22 µm filter *
  5. Microloader (Eppendorf, catalog number: 5242956003 )
  6. Glass capillary for mouth pipettes (Drummond Scientific, catalog number: 1-000-0500 )
  7. Glass capillary for holding pipettes (Sutter Instrument, catalog number: B100-75-10 )
  8. Glass capillary for injection pipettes (World Precision Instruments, catalog number: TW100F-4 )
  9. Male and female C57BL/6 x DBA/2 hybrid (B6D2F1) or other strains (for recipient zygotes)
  10. Male and female ICR (for preparation of pseudopregnant mice)
  11. Cas9 plasmids (available from Addgene)
    1. Nickase-type of Cas9 (nCas9) (Addgene, catalog number: 41816 )
    2. FokI domain-fused catalytically-inactive (fCas9) (Addgene, catalog number: 52970 )
  12. PrimeSTAR MAX (Takara Bio, catalog number: R045A ) or other PCR polymerases
  13. Qiaquick PCR Purification Kit (QIAGEN, catalog number: 28104 ) or equivalent
  14. AgeI*
  15. mMESSAGE/mMACHINE T7 Transcription Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM1344 )
  16. MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM1908 )
  17. Pregnant mare serum gonadotropin (PMSG) (ASKA Animal Health, Serotropin®, catalog number: 879412 )
  18. Human chronic gonadotropin (hCG) (ASKA Animal Health, catalog number: Gonatropin 3000 )
  19. Hyaluronidase (Sigma-Aldrich, catalog number: H4272 )
  20. KSOM medium (ARK resource)
  21. Dichlorodimethylsilane (Tokyo Chemical Industry, catalog number: D0358 )
  22. ExoSAP-IT (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 78200.200. UL )
  23. Sodium chloride (NaCl) (Wako Pure Chemical Industries, catalog number: 191-01665 )
  24. Potassium chloride (KCl) (Nacalai Tesque, catalog number: 28514-75 )
  25. Calcium chloride (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C7902 )
  26. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  27. Magnesium sulfate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: M2773 )
  28. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
  29. HEPES (DOJINDO, catalog number: 342-01375 )
  30. Sodium DL-lactate (Sigma-Aldrich, catalog number: L7900 )
  31. Sodium pyruvate (Sigma-Aldrich, catalog number: P2256 )
  32. D-(+)-glucose (Sigma-Aldrich, catalog number: G7528 )
  33. Polyvinyl alcohol (Sigma-Aldrich, catalog number: P8136 )
  34. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  35. Phenol red (Sigma-Aldrich, catalog number: P0290 )
  36. Sodium hydroxide (NaOH) (Sigma-Aldrich)
  37. Paraffin liquid (Nacalai Tesque, catalog number: 26137-85 )
  38. BIOTAQ (Bioline, catalog number: BIO-21040 ) or other PCR polymerases
  39. M2 medium (see Recipes)

*Note: No particular preference.

Equipment

  1. Spectrophotometer (Thermo Fisher Scientific, model: NanoDrop ND-1000 )
  2. Capillary puller (Sutter Instrument, model: P-1000 )
  3. Microforge (NARISHIGE, model: MF-900 )
  4. Micro centrifuge*
  5. Thermal cycler*
  6. Heating block*
  7. Upright microscope*
  8. Micromanipulator (NARISHIGE, model: NT-88-V3 )
  9. Injectors (NARISHIGE, models: IM-11-2 and IM-9B )
  10. FemtoJet (Eppendorf, model: FemtoJet® Express )
  11. CO2 incubator (37 °C, 5% CO2, and 95% air condition)

*Note: No particular preference.

Procedure

  1. crRNA design
    1. To select target sequences of crRNAs, a list of possible sgRNAs are required first. We recommend using online web tools such as CRISPR design tool (http://crispr.mit.edu/) or CRISPRdirect (https://crispr.dbcls.jp/) for this purpose. Choose a pair of sgRNAs with the following criteria: spacer distance is 4-20 base pairs (bp) for nCas9 or is 14-19 bp for fCas9 (Figure 1) and the pair of sgRNAs must be located on opposite strands of genomic DNA (Ran et al., 2013; Hara et al., 2015).
    2. Design crRNA sequences by removing the protospacer adjacent motif (PAM) and replacing T (thymine) with U (uracil) from the sequences of a pair of sgRNAs as shown in Figure 1.
    3. Have selected crRNAs (42 nt) and tracrRNA (69 nt) chemically synthesized and purified by HPLC. We adjust the concentration of crRNAs and tracrRNA to 1 µg/µl.


      Figure 1. Design of crRNA target sites for modified Cas9s. (Top) crRNA target sites are underlined in genomic DNA. Both the sense and antisense strands of a portion of the genomic sequence at the intronic region of the Bcr gene are shown. (Middle) The two target sites for crRNAs are underlined. Protospacer adjacent motif (PAM) sequences (NGG) appear in bold letters. The spacer sequence is indicated by a gray dashed double-headed arrow. (Bottom) crRNA sequence (42 nt) for each target site and the sequence of tracrRNA (69 nt) are shown. The crRNA-specific sequence (22 nt), derived from Jinek et al. (2013), is shown in blue. Uracil is shown in red.

  2. Preparation of Cas9 mRNA
    1. Preparation of DNA templates for in vitro transcription
      1. Synthesize Cas9 mRNA in an in vitro transcription reaction using T7 RNA polymerase. Note, the plasmid that contains fCas9 has a T7 promoter sequence, but the plasmid that contains nCas9 does not (Figure 2). To add the T7 promoter sequence to the 5’ terminal of nCas9, amplify nCas9 by PCR with the following primers:
        T7-Cas9-F: TAATACGACTCACTATAGGGAGAATGGACAAGAAGTACTCCATTGG (underline: T7 promoter sequence)
        Cas9-R: TCACACCTTCCTCTTCTTC
        Check the PCR product (4,163 bp) by agarose gel electrophoresis.
      2. Purify PCR products with the Qiaquick PCR Purification Kit (according to the manufacturer’s instructions). The concentration of purified products can be determined using a spectrophotometer.
      3. Digest the plasmid containing fCas9 with AgeI and purify the products with the Qiaquick PCR Purification Kit (according to the manufacturer’s instructions).


        Figure 2. Preparation of Cas9 mRNA. Schematic representations of Cas9 mRNA synthesis. Black lines and arrows show backbone vector and primers respectively. Cytomegalovirus promoter (pCMV), nCas9 and fCas9 are boxed. T7 promoter sequences are depicted with green lines. (Left) Since nCas9 vector (Addgene #41816) does not contain T7 promoter, T7 promoter is added to the nCas9 coding sequence by PCR using T7-Cas9-F/Cas9-R primers and the plasmid as a template. PCR products are then subjected to in vitro transcription reaction. (Right) fCas9 vector (Addgene #52970) includes T7 promoter, which can be used for in vitro transcription reaction after linearization of the plasmid by AgeI digestion.

    2. In vitro transcription reactions are performed using the mMESSAGE/mMACHINE T7 Transcription Kit (according to the manufacturer’s instructions) with 2 µg of the PCR product or the linearized plasmid as template. After TURBO DNase treatment (included in the mMESSAGE/mMACHINE T7 Transcription Kit), the transcribed RNA products are purified with the MEGAclear Transcription Clean-Up Kit. We adjust the concentration of the mRNA product to 1 µg/µl. 
    3. For microinjection, RNAs are mixed as follows:
      1 µg/µl of crRNA1 and crRNA2 2.84 µl each
      1 µg/µl of tracrRNA 9.32 µl
      1 µg/µl of nCas9 or fCas9 mRNA 7.5 µl
      RNase-free water 7.5 µl
      Total volume = 30 µl
      Notes: 
      1. Final concentrations of crRNA1, crRNA2, tracrRNA and Cas9 mRNA are 94.7, 94.7, 310.7 and 250 ng/µl, respectively.
      2. The molar ratios of crRNAs and tracrRNA are adjusted to be equal in the mixture. Molar ratios were calculated by dividing RNA concentration by RNA length. Molar ratios of crRNA1, crRNA2 and tracrRNA are 94.7 ng/µl/42 nt (= 2.25), 94.7 ng/µl/42 nt (= 2.25) and 310.7 ng/µl/69 nt (= 4.5), respectively.
      3. Although the RNA mixture can be stored for several months at -80 °C, we recommend that the RNA mixture be prepared fresh each time.

  3. Collection of fertilized eggs
    1. On Day 1, induce superovulation in adult female mice by injecting them with 5 IU PMSG intraperitoneally. Forty-eight hours later (Day 3) inject the female mice with 5 IU hCG, then cross with adult male mice.
    2. On Day 4, sacrifice the female mice that were crossed on Day 3 and collect fertilized eggs from their oviducts. To remove cumulus cells, treat the zygotes with 0.3 mg/ml of hyaluronidase in M2 medium (see Recipes) at 37 °C for 1-5 min.
    3. Pick zygotes using mouth pipettes and then wash several times by pipetting in KSOM medium.

  4. Preparation of holding and injection pipettes
    1. Glass capillaries are drawn out using a capillary puller.
    2. Holding and injection pipettes are prepared from drawn-out pipettes using a microforge.
    3. Before injection, centrifuge the RNA mixture at 12,000 x g for 10 min. Fill the injection pipettes with 3-5 µl of the supernatant of the RNA mixture by microloader.
    4. Coat the outside of the RNA-filled injection pipettes with dichlorodimethylsilane.

  5. Microinjection
    1. Put the fertilized eggs in a drop of M2 medium and insert the injection pipette into the cytoplasm of zygote. Inject the RNA mixture (1-5 pl) into the cytoplasm. The amount of the injected solution can be controlled using FemtoJet.
    2. Pool the injected eggs in M2 medium. Wash the zygotes several times with KSOM medium. Incubate in a drop of KSOM drop at 37 °C until next day.

  6. 2-cell embryo transfer
    1. Obtain pseudopregnant mice by crossing adult ICR females with vasectomized male mice on the same day of microinjection (Day 4).
    2. On the morning of Day 5, check ICR females for a vaginal plug. If the ICR female has a vaginal plug, then it is considered to be pseudopregnant.
    3. Isolate injected embryos that have reached the 2-cell stage by picking them up in a minimal volume of KSOM medium (10-18 embryos). Transfer these embryos into either oviduct an anesthetized, pseudopregnant female mouse.
    4. Nineteen days after transplantation, newborn mice should be obtained.
    Note: Procedure C to Procedure F is a standard procedure of embryo treatment that is detailed in Nagy et al. (2003).

  7. Genotyping the founder mice by PCR direct sequencing
    1. Isolate genomic DNA from a tail tip using a standard procedure (Nagy et al., 2003).
    2. Amplify the region of interest using primers designed around crRNA target sequences by performing PCR with the genomic DNA and primers spanning the crRNAs target sequences. Treat the PCR products with ExoSAP-IT (or Qiaquick PCR purification Kit) and sequence with either primer used for PCR.

Data analysis

When a mutation is introduced heterozygously by crRNA/tracrRNA/Cas9, overlapped double peaks will be observed between the two target sites (see Figure 3). If a homozygous or mosaic mutation is introduced, then a single or more than two peaks will be observed, respectively. In addition to sequencing the direct PCR product, one can also clone the PCR product and sequence the plasmid. Note that there is a possibility of having a mosaic mutation even when a single peak is observed in the sequence at this step. It is recommended that the sequence at the site of mutation as well as any relevant phenotypes be analyzed in the F1 generation as well as or later generations, if possible.
Using this method, from our experience, approximately 30-40% and 30-60% of founder pups obtained after crRNA/tracrRAN/nCas9 and crRNA/tracrRNA/fCas9 injection, respectively, contained mutations at least one allele (Terao et al., 2016).


Figure 3. Example of genotyping analysis. Electropherograms of PCR products amplified from wild-type mice (top), mice carrying a mutation in a single allele (middle), and mice carrying a mutation in both alleles (bottom). Each target site of crRNA is underlined with a solid black line. If the target site is underlined with a dashed line, then it indicates that the target site is broken (see Biallelic mutation, bottom). The dashed arrow indicates a point of mutation.

Recipes

  1. M2 medium
    94.66 mM NaCl
    4.78 mM KCl
    1.71 mM CaCl2
    1.19 mM KH2PO4
    1.19 mM MgSO4
    4.15 mM NaHCO3
    20.85 mM HEPES
    23.28 mM sodium DL-lactate
    0.33 mM sodium pyruvate
    5.56 mM D-(+)-glucose
    0.01% (w/v) polyvinyl alcohol
    0.5% (v/v) penicillin-streptomycin
    0.02% (v/v) phenol red
    Adjust the pH to 7.4 with 5-10 N NaOH
    Filtration with a 0.22 µm filter

Acknowledgments

This work was supported in part by a grant from the National Center for Child Health and Development, Grant Number 24-3 to S.T. This protocol is developed based on our previous work published in Exp Anim (Terao et al., 2016).

References

  1. Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A. and Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121): 819-823.
  2. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A. and Charpentier, E. (2012). A Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096): 816-821.
  3. Hara, S., Tamano, M., Yamashita, S., Kato, T., Saito, T., Sakuma, T., Yamamoto, T., Inui, M. and Takada, S. (2015). Generation of mutant mice via the CRISPR/Cas9 system using FokI-dCas9. Sci Rep 5: 11221.
  4. 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(6121): 823-826.
  5. Nagy, A., Gertsenstein, M., Vintersten, K. and Behringer, R. (2003). Manipulating the mouse embryo: A laboratory manual, 3rd edition. Cold Spring Harbor Laboratory Press.
  6. Ran, F. A., Hsu, P. D., Lin, C. Y., Gootenberg, J. S., Konermann, S., Trevino, A. E., Scott, D. A., Inoue, A., Matoba, S., Zhang, Y. and Zhang, F. (2013). Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154(6): 1380-1389.
  7. Terao, M., Tamano, M., Hara, S., Kato, T., Kinoshita, M. and Takada, S. (2016). Utilization of the CRISPR/Cas9 system for the efficient production of mutant mice using crRNA/tracrRNA with Cas9 nickase and FokI-dCas9. Exp Anim 65(3): 275-283.

简介

聚类规则间隔短回文重复(CRISPR)/ CRISPR相关蛋白9(Cas9)系统是使用最广泛的基因组编辑工具。一个常见的CRISPR / Cas9系统由两个组成部分组成:单导RNA(sgRNA)和Cas9。在特定靶序列引入双链断裂(DSB)需要两种成分。该系统的一个缺点是实验室中sgRNA的生产是费力的,因为它需要在体外​​转录反应和sgRNA纯化之间克隆sgRNA序列。通过sgRNA靶向Cas9活性的替代方案是用两种小RNA:CRISPR RNA(crRNA)和反式激活性crRNA(tracrRNA)进行靶向。这两种小RNA可以化学合成,这使得与sgRNA相比,这些RNA的产生不那么困难。 CRISPR / Cas9系统的另一个缺点是已经报告了脱靶效应。然而,已经开发了改进形式的Cas9以最小化离靶效应。例如,仅当两个引导RNA在规定的距离内结合相对的链时,切口酶型Cas9(nCas9)和FokI结构域融合的催化无活性的Cas9(FokI-dCas9; fCas9)才诱导DSB。在本协议中,我们描述了使用结合crRNA,tracrRNA和Cas9修饰形式的CRISPR / Cas9系统来生产突变小鼠的实验系统。该方法不仅有利于制备用于基因组编辑系统的试剂,而且可以降低脱靶效应的风险。

背景 聚类规则间隔短回文重复(CRISPR)/ CRISPR相关蛋白9(Cas9)系统是一种有效的基因组编辑工具。在细菌中,CRISPR / Cas9作为适应性免疫系统。它由两个小RNA,即CRISPR RNA(crRNA)和反式激活crRNA(tracrRNA)和Cas9 DNA核酸酶组成,其中消化靶向DNA(Jinek等人,2013)。几个组织已经建立了CRISPR / Cas9系统作为在许多细胞类型中引入突变的工具(Cong等人,2013; Mali等人,2013)。当Cas9核酸酶靶向基因组DNA时,它切割DNA,导致通过非同源末端连接(NHEJ)修复的病变或同源DNA重组。由于NHEJ可能是一个容易出错的机制,当通过这种机制修复DNA时,可以将突变引入基因组。 CRISPR / Cas9系统可用于通过将Cas9和单导向RNA(sgRNA)显微注射入受精卵来编辑小鼠的基因组。尽管sgRNA已被广泛用于成功,但sgRNA的产生是费力的,因为sgRNA必须从DNA寡聚体克隆,然后在体外转录。使用crRNA和tracrRNA的系统可以消除制备sgRNA的大量劳力,因为crRNA和tracrRNA的长度足够小以进行化学合成。因此,不是用sgRNA注射Cas9,通过用crRNA和tracrRNA(crRNA / tracrRNA)显微注射Cas9可以获得突变小鼠。使用CRISPR / Cas9的一个缺点是引导RNA / Cas9复合物可以产生非目标突变,这是在靶RNA序列与引导RNA的靶序列具有相似序列的位点发生的非预期突变。通过引入Cas9修饰形式,如Cas9(nCas9)和FokI结构域融合的催化无活性Cas9(FokI-dCas9; fCas9)的切口酶类型,可以降低诱导靶外突变的风险。这些Cas9形式只有当两个sgRNA以相互间距离相对的两条链结合时,才能切割目标DNA(Ran等人,2013; Hara等人,2015)。最近,我们通过将化学合成的crRNA / tracrRNA显微注射到受精卵中成功产生突变小鼠(Terao等人,2016)。该协议减少了制备靶向RNA的时间和劳动力,并且可以降低脱靶效应的风险。

关键字:CRISPR / Cas9,  crRNA / tracrRNA,  nCas9,  fCas9, 突变小鼠

材料和试剂

  1. 35毫米盘*
  2. 60毫米盘*
  3. 口吸移液管*
  4. 0.22μm过滤器*
  5. Microloader(Eppendorf,目录号:5242956003)
  6. 用于吸液管的玻璃毛细管(Drummond Scientific,目录号:1-000-0500)
  7. 用于保持移液器的玻璃毛细管(Sutter Instrument,目录号:B100-75-10)
  8. 用于注射移液管的玻璃毛细管(World Precision Instruments,目录号:TW100F-4)
  9. 雄性和雌性C57BL / 6×DBA / 2杂种(B6D2F1)或其他菌株(用于接受体合子)
  10. 男性和女性ICR(用于制备假孕小鼠)
  11. Cas9质粒(可从Addgene获得)
    1. Cas9(nCas9)的茜素型(Addgene,目录号:41816)
    2. FokI域融合催化无活性(fCas9)(Addgene,目录号:52970)
  12. PrimeSTAR MAX(Takara Bio,目录号:R045A)或其他PCR聚合酶
  13. Qiaquick PCR纯化试剂盒(QIAGEN,目录号:28104)或等同物
  14. 年龄 I *
  15. mMESSAGE / mMACHINE T7转录试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:AM1344)
  16. MEGAclear转录清除试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:AM1908)
  17. 怀孕母马血清促性腺激素(PMSG)(ASKA Animal Health,Serotropin ,目录号:879412)
  18. 人慢性促性腺激素(hCG)(ASKA Animal Health,目录号:Gonatropin 3000)
  19. 透明质酸酶(Sigma-Aldrich,目录号:H4272)
  20. KSOM媒体(ARK资源)
  21. 二氯二甲基硅烷(Tokyo Chemical Industry,目录号:D0358)
  22. ExoSAP-IT(Thermo Fisher Scientific,Applied Biosystems TM,目录号:78200.200 UL)
  23. 氯化钠(NaCl)(Wako Pure Chemical Industries,目录号:191-01665)
  24. 氯化钾(KCl)(Nacalai Tesque,目录号:28514-75)
  25. 氯化钙(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C7902)
  26. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  27. 硫酸镁(MgSO 4·7H 2 O)(Sigma-Aldrich,目录号:M2773)
  28. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S5761)
  29. HEPES(DOJINDO,目录号:342-01375)
  30. DL-乳酸钠(Sigma-Aldrich,目录号:L7900)
  31. 丙酮酸钠(Sigma-Aldrich,目录号:P2256)
  32. D-(+) - 葡萄糖(Sigma-Aldrich,目录号:G7528)
  33. 聚乙烯醇(Sigma-Aldrich,目录号:P8136)
  34. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  35. 苯酚红(Sigma-Aldrich,目录号:P0290)
  36. 氢氧化钠(NaOH)(Sigma-Aldrich)
  37. 石蜡液(Nacalai Tesque,目录号:26137-85)
  38. BIOTAQ(Bioline,目录号:BIO-21040)或其他PCR聚合酶
  39. M2介质(见配方)

*注意:没有特别的偏好。

设备

  1. 分光光度计(Thermo Fisher Scientific,型号:NanoDrop ND-1000)
  2. 毛细管拉拔器(Sutter Instrument,型号:P-1000)
  3. Microforge(NARISHIGE,型号:MF-900)
  4. 微型离心机*
  5. 热循环仪*
  6. 加热块*
  7. 立式显微镜*
  8. 微操纵器(NARISHIGE,型号:NT-88-V3)
  9. 注射器(NARISHIGE,型号:IM-11-2和IM-9B)
  10. FemtoJet(Eppendorf,型号:FemtoJet ® Express)
  11. CO 2培养箱(37℃,5%CO 2和95%空气条件)

*注意:没有特别的偏好。

程序

  1. crRNA设计
    1. 为了选择crRNA的靶序列,首先需要可能的sgRNA的列表。我们建议使用在线网络工具,如CRISPR设计工具( http://crispr.mit.edu / )或CRISPRdirect( https://crispr.dbcls.jp/)为此目的。 选择一对具有以下标准的sgRNA:间隔距离对于nCas9为4-20个碱基对(bp),对于fCas9为14-19bp(图1),并且该对sgRNA必须位于基因组DNA的相对链上Ran等人,2013; Hara等人,2015)。
    2. 通过从一对sgRNA的序列中除去原始相邻基序(PAM)并用U(尿嘧啶)代替T(胸腺嘧啶)来设计crRNA序列,如图1所示。
    3. 选择通过HPLC化学合成和纯化的crRNA(42nt)和tracrRNA(69nt)。我们将crRNA和tracrRNA的浓度调整为1μg/μl

      图1.修饰的Cas9的crRNA靶位点的设计(顶部)crRNA靶位点在基因组DNA中被加下划线。显示了在Bcr基因的内含子区域的一部分基因组序列的有义链和反义链。 (中)crRNA的两个靶位点加下划线。 Protospacer相邻基序(PAM)序列(NGG)以粗体字出现。间隔序列由灰色虚线双头箭头表示。 (底部)crRNA序列(42nt)和tracrRNA(69nt)的序列。来自Jinek等人的crRNA特异性序列(22nt)。 (2013),显示为蓝色。尿嘧啶显示为红色。

  2. Cas9 mRNA的制备
    1. 制备用于体外转录的DNA模板
      1. 使用T7 RNA聚合酶在体外转录反应中合成Cas9 mRNA。注意,含有fCas9的质粒具有T7启动子序列,但是含有nCas9的质粒不是(图2)。为了将T7启动子序列添加到nCas9的5'末端,用以下引物通过PCR扩增nCas9:
        T7-Cas9-F:TAATACGACTCACTATAGGGAGA ATGGACAAGAAGTACTCCATTGG(下划线:T7启动子序列)
        Cas9-R:TCACACCTTCCTCTTCTTC
        通过琼脂糖凝胶电泳检查PCR产物(4,163bp)
      2. 使用Qiaquick PCR纯化试剂盒(根据制造商的说明书)纯化PCR产物。纯化产物的浓度可以用分光光度计测定
      3. 将含有fCas9的质粒用“AgeI”消化,并用Qiaquick PCR Purification Kit(根据制造商的说明书)纯化产物。


        图2. Cas9 mRNA的制备 Cas9 mRNA合成的示意图。黑线和箭头分别显示骨架载体和引物。巨细胞病毒启动子(pCMV),nCas9和fCas9是盒装的。用绿线描绘T7启动子序列。 (左)由于nCas9载体(Addgene#41816)不含有T7启动子,所以通过使用T7-Cas9-F / Cas9-R引物和质粒作为模板的PCR,将T7启动子加入到nCas9编码序列中。然后将PCR产物进行体外转录反应。 (右)fCas9载体(Addgene#52970)包括T7启动子,其可以通过 I消化在质粒线性化后用于体外转录反应。 >
    2. 使用mMESSAGE / mMACHINE T7转录试剂盒(根据制造商的说明书),使用2μgPCR产物或线性化质粒作为模板进行体外转录反应。在TURBO DNA酶处理(包括在mMESSAGE / mMACHINE T7转录试剂盒中)之后,转录的RNA产物用MEGAclear Transcription Clean-Up Kit纯化。我们将mRNA产物的浓度调整为1μg/μl。 
    3. 对于显微注射,RNA如下混合:
      1μg/μl的crRNA1和crRNA22.84μl,每个
      1μg/μltracrRNA9.32μl
      1μg/μlnCas9或fCas9 mRNA 7.5μl
      无RNase的水7.5μl
      总体积= 30μl
      注意:
      1. crRNA1,crRNA2,tracrRNA和Cas9 mRNA的最终浓度分别为94.7,94.7,310.7和250ng /μl。
      2. crRNA和tracrRNA的摩尔比在混合物中调整为相等。通过将RNA浓度除以RNA长度计算摩尔比。 crRNA1,crRNA2和tracrRNA的摩尔比分别为94.7 ng /μl/ 42 nt(= 2.25),94.7 ng /μl/ 42 nt(= 2.25)和310.7 ng /μl/ 69 nt(= 4.5) >
      3. 尽管RNA混合物可以在-80℃下储存几个月,但我们建议每次新鲜制备RNA混合物。

  3. 收集受精卵
    1. 在第1天,通过用5IU PMSG腹膜内注射成年雌性小鼠诱导超排卵。 48小时后(第3天)用5 IU hCG注射雌性小鼠,然后与成年雄性小鼠交叉。
    2. 在第4天,牺牲在第3天穿过的雌性小鼠,并从输卵管收集受精卵。为了去除卵丘细胞,在M2培养基(参见食谱)中用0.3mg / ml透明质酸酶处理合子,在37℃下1-5分钟。
    3. 使用吸液管选择合子,然后通过KSOM培养基中的移液冲洗数次。

  4. 保持和注射移液管的准备
    1. 玻璃毛细管用毛细管牵引器拉出。
    2. 保持和注射移液器由使用微型锻造的拉出移液器制备。
    3. 注射前,以12,000 x g离心RNA混合物10分钟。通过微型加载机将注射移液管装入3-5μlRNA混合物的上清液。
    4. 用二氯二甲基硅烷涂覆RNA填充的注射移液管外面。

  5. 显微注射
    1. 将受精卵放入一滴M2培养基中,并将注射移液管插入受精卵细胞质。将RNA混合物(1-5μl)注入细胞质。注射溶液的量可以使用FemtoJet进行控制。
    2. 在M2培养基中灌注注射的卵。用KSOM培养基洗涤受精卵几次。在37°C下降到KSOM下降至第二天。

  6. 2细胞胚胎移植
    1. 在显微注射的同一天,通过成年ICR女性与输精管切除的雄性小鼠相交获得假孕小鼠(第4天)。
    2. 在第5天的早晨,检查ICR女性的阴道塞。如果ICR女性有阴道塞,则认为是假孕
    3. 通过在最小体积的KSOM培养基(10-18胚胎)中接种来分离已经达到2细胞阶段的注射的胚胎。将这些胚胎转移到输卵管麻醉,假孕雌性小鼠中
    4. 移植后十九天,应获得新生儿。
    注意:程序C至程序F是Nagy等人详细描述的胚胎治疗的标准程序。 (2003)。

  7. 通过PCR直接测序法对创始人小鼠进行基因分型
    1. 使用标准程序从尾尖分离基因组DNA(Nagy等人,2003)。
    2. 使用围绕crRNA靶序列设计的引物扩增感兴趣的区域,通过用基因组DNA和跨越crRNA靶序列的引物进行PCR。用ExoSAP-IT(或Qiaquick PCR纯化试剂盒)和使用PCR引物的序列处理PCR产物。

数据分析

当通过crRNA / tracrRNA / Cas9杂合引入突变时,将在两个靶位点之间观察到重叠的双峰(参见图3)。如果引入纯合或镶嵌突变,则分别观察到单个或多于两个峰。除了对直接PCR产物进行测序之外,还可以克隆PCR产物并对质粒进行测序。注意,即使在该步骤中的序列中观察到单个峰,也存在镶嵌突变的可能性。建议在可能的情况下,在F 1/2代以及后代中分析突变位点的序列以及任何相关表型。
使用这种方法,根据我们的经验,分别在crRNA / tracrRAN / nCas9和crRNA / tracrRNA / fCas9注射后获得的大约30-40%和30-60%的创始人幼崽分别含有至少一个等位基因的突变(Terao et al。 ,2016)。


图3.基因分型分析实例从野生型小鼠(上)扩增的PCR产物的电泳图,在单个等位基因(中间)中携带突变的小鼠,以及在两个等位基因中携带突变的小鼠底部)。 crRNA的每个靶位点用实线黑线划线。如果目标网站用虚线加下划线,则表示目标网站被破坏(参见Biallelic突变,底部)。虚线箭头表示突变点。

食谱

  1. M2媒体
    94.66 mM NaCl
    4.78 mM KCl
    1.71mM CaCl 2
    1.19mM KH 2 PO 4
    1.19mM MgSO 4
    4.15mM NaHCO 3
    20.85 mM HEPES
    23.28mM DL-乳酸钠
    0.33mM丙酮酸钠
    5.56mM D-(+) - 葡萄糖 0.01%(w / v)聚乙烯醇
    0.5%(v / v)青霉素 - 链霉素
    0.02%(v / v)酚红
    用5-10N NaOH调节pH至7.4 用0.22μm过滤器过滤

致谢

这项工作部分得到了国家儿童健康与发展中心赠款24-3号给S.T.的资助。该协议是根据我们以前在Exp Anim (Terao等人,2016年)中发表的工作开发的。

参考

  1. Cong,L.,Ran,FA,Cox,D.,Lin,S.,Barretto,R.,Habib,N.,Hsu,PD,Wu,X.,Jiang,W.,Marraffini,LA and Zhang,F 。(2013)。使用CRISPR / Cas的多重基因组工程系统。 科学 339(6121):819-823。
  2. Jinek,M.,Chylinski,K.,Fonfara,I.,Hauer,M.,Doudna,JA和Charpentier,E.(2012)。  可编程双RNA引导的DNA内切核酸酶在适应性细菌免疫中的应用 科学 337(6096) :816-821。
  3. Hara,S.,Tamano,M.,Yamashita,S.,Kato,T.,Saito,T.,Sakuma,T.,Yamamoto,T.,Inui,M.and Takada,S。(2015) 使用FokI-dCas9通过CRISPR / Cas9系统产生突变体小鼠。 Sci Rep 5:11221.
  4. Mali,P.,Yang,L.,Esvelt,KM,Aach,J.,Guell,M.,DiCarlo,JE,Norville,JE and Church,GM(2013)。  通过Cas9进行RNA指导的人类基因组工程 科学 339( 6121):823-826。
  5. Nagy,A.,Gertsenstein,M.,Vintersten,K.和Behringer,R。(2003)。操作小鼠胚胎:实验室手册,第3版冷泉港实验室出版社
  6. Ran,FA,Hsu,PD,Lin,CY,Gootenberg,JS,Konermann,S.,Trevino,AE,Scott,DA,Inoue,A.,Matoba,S.,Zhang,Y.and Zhang, )。 RNA引导CRISPR Cas9的双切口增强基因组编辑特异性。 细胞 154(6):1380-1389。
  7. Terao,M.,Tamano,M.,Hara,S.,Kato,T.,Kinoshita,M.and Takada,S。(2016)。< a class =“ke-insertfile”href =“http: /www.ncbi.nlm.nih.gov/pubmed/26972821“target =”_ blank“>使用CRISPR / Cas9系统,利用具有Cas9切口酶和FokI-dCas9的crRNA / tracrRNA有效生产突变小鼠。 Exp Anim 65(3):275-283。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hara, S., Terao, M. and Takada, S. (2017). A Protocol for Production of Mutant Mice Using Chemically Synthesized crRNA/tracrRNA with Cas9 Nickase and FokI-dCas9. Bio-protocol 7(11): e2340. DOI: 10.21769/BioProtoc.2340.
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