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Dec 2018

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Construction of Viral Vectors for Cell Type-specific CRISPR Gene Editing in the Adult Mouse Brain
用于成年小鼠脑组织的细胞特异性CRISPR基因编辑的病毒载体的构建   

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Abstract

Recently developed gene editing technologies based on engineered CRISPR/Cas9 systems enables researchers to disrupt genes in a cell type-specific manner in the adult mouse brain. Using these technologies, we recently showed that the dopamine beta-hydroxylase gene in Locus Coeruleus (LC) norepinephrine neurons plays a vital role in the maintenance of wakefulness. Our method consists of four steps, (1) crossing Cre-dependent spCas9 knockin mice with a Cre-driver mouse line to express spCas9 in the target neural populations, (2) cloning of sgRNA, (3) construction of an AAV (adeno associated virus) vector expressing dual sgRNA, and (4) virus packaging and stereotaxic injection of the virus into the target brain area. Here, we describe a detailed protocol of AAV vector construction for cell type-specific CRISPR gene editing in the adult mouse brain. The method adopts a dual-sgRNA strategy for efficient disruption of the target gene. At first, a few different sgRNAs targeting the same gene are cloned into a plasmid expressing spCas9. After evaluation of the sgRNAs by a T7 endonuclease assay, the two most efficient sgRNAs are cloned in tandem into an AAV vector using the Gibson Assembly method.

Keywords: CRISPR/Cas9 (CRISPR/Cas9), AAV (AAV), Adult mouse brain (成年小鼠脑组织), Gibson Assembly (Gibson 装配), dual-sgRNA (双sgRNA)

Background

Interrogation of gene functions in specific cell subtypes in the brain remains a challenge. The conventional techniques such as RNAi-based methods lack cellular specificity and efficiency. On the other hand, recently developed gene editing techniques using the CRISPR/Cas9 system allows researchers efficient gene disruption in a cell type-specific manner. A research group led by Feng Zhang showed the disruption of multiple genes in the adult mouse brain by AAV-mediated delivery of sgRNA (Swiech et al., 2015). Also, the group established cre-dependent spCas9 knockin mice (Platt et al., 2014). In a recent study, we used an AAV vector carrying dual sgRNA to improve the efficiency of bi-allelic gene targeting (Yamaguchi et al., 2018). We here describe a protocol for the construction of dual sgRNA AAV vector (Figure 1).


Figure 1. Schematic representation of all the procedure. Our method consists of four steps: (1) crossing Cre-dependent spCas9 knockin mice with a Cre-driver mouse line to express spCas9 in the target neural populations, (2) cloning of sgRNA into the spCas9 expressing plasmid, (3) construction of an AAV vector tandemly expressing dual sgRNA, and (4) AAV packaging and stereotaxic viral injection into the target brain area.

Materials and Reagents

  1. Pipette tips
  2. 24-well plate (coated) (Thermo Fisher Scientific, catalog number: 142475)
  3. 1 ml plastic tube
  4. NEB10-beta Competent E. coli (New England Biolabs, catalog number: C3019)
  5. pX458 (Addgene, catalog number: 48138)
  6. pAAV EF1α DIO mCherry (Addgene, catalog number: 20299)
  7. BbsI-HF (New England Biolabs, catalog number: R3539)
  8. MluI-HF (New England Biolabs, catalog number: R3198)
  9. DpnI (New England Biolabs, catalog number: R0176)
  10. Alkaline Phosphatase, Calf Intestinal (CIP) (New England Biolabs, catalog number: M0290)
  11. Guide oligonucleotides (see Procedure A)
  12. Quick ligation kit (New England Biolabs, catalog number: M2200)
  13. Gibson Assembly Master Mix (New England Biolabs, catalog number: E2611)
  14. PrimeSTAR HS DNA polymerase (Takara, catalog number: R010)
  15. dNTP Mix 10 mM each (the mix of 10 mM of dTTP, dCTP, dGTP and dATP) (GenScript, catalog number: C01689)
  16. QIAquick Gel Extraction kit (QIAGEN, catalog number: 28704)
  17. QIAprep Spin Miniprep kit (QIAGEN, catalog number: 27104)
  18. Tris-EDTA, (1x Solution) (Fisher BioReagents, catalog number: BP2473)
  19. LB Broth EZMix Powder (Sigma, catalog number: L7658)
  20. LB Agar (Fisher BioReagents, catalog number: BP1425)
  21. Ampicillin (Gemini Bio-Products, catalog number: 400-130P)
  22. Primer to verify the cloning of sgRNA into pX458: 5’-gactatcatatgcttaccgt-3’
  23. Primer to verify the tandem cloning of sgRNAs into pAAV: 5’-actgacgggcaccggagcca-3’
  24. Primers for Gibson Assembly:
    Primer S1: 5’-taggggttcctgcggccgcacgcgtgagggcctatttc-3’
    Primer AS1: 5’-ataggccctctctagaaaaaaagcaccgactc-3’
    Primer S2: 5’-tttttctagagagggcctatttcccatg-3’
    Primer AS2: 5’-atccatctttgcaaagcttacgcgtaaaaaagcaccgac-3’

    Optional (see Procedure C)

  25. NIH3T3 cell (ATCC, catalog number: CRL-1658)
  26. AAV vector (pX458-sgRNA or pX458 empty as negative control )
  27. Primers: You can design the primers to amplify the target exons by CHOPCHOP (see Procedure C)
  28. PrimeSTAR HS DNA polymerase
  29. FuGENE6 Transfection Reagent (Promega, catalog number: E2693)
  30. DMEM (1x) (Gibco, catalog number: 11995-065)
  31. Fetal Bovine Serum (Corning, catalog number: 35-015-CV)
  32. Penicillin/Streptomycin (Gibco, catalog number: 15070-063)
  33. PBS, pH 7.4 (Gibco, catalog number: 10010-023)
  34. TrypLE Select (1x) (Gibco, catalog number: 12563-011)
  35. QuickExtract DNA Extraction Solution (Epicentre, catalog number: QE0905T)
  36. T7 Endonuclease I (New England Biolabs, catalog number: M0302)

Equipment

  1. Pipettes (Gilson, Pipetman classic P10, P20, P200)
  2. Centrifuge (Eppendorf, Centrifuge 5424)
  3. Incubator (VWR, incubating 5000IR orbital shaker)
  4. Thermocycler (Applied Biosystems, GeneAmp PCR system 2700)
  5. Electrophoresis chamber (Labnet, ENDURO GEL XL)
  6. (Optional) CO2 Incubator (Binder, CB 170)
  7. (Optional) Clean bench (Labconco, Purifier classII biosafety cabinet)
  8. (Optional) Centrifugation (Thermo Scientific, Soryall legend XTR)

Procedure

  1. Design of sgRNA
    Single guide RNAs (sgRNAs) with high efficiency and specificity can be designed by using gRNA design webtools. We recommend CHOPCHOP (http://chopchop.cbu.uib.no) because of its user-friendly interface (Labun et al., 2016). Also, you may find the guide design resources on the website of Feng Zhang’s lab (https://zlab.bio/guide-design-resources). The sgRNA should target early exons (near transcriptional start site) or essential exons of gene function. We usually choose 4 to 6 target sites on early exons which do not have the start codon because frameshift mutations near the start codon can lead to translation from other in-frame ATGs, resulting in the expression of a functional protein. If the target sequence is 5’-CGCAGGACCAGACCCCATAATGG-3’ [Protospacer adjacent motif (PAM) is labeled in blue], synthesize 5’-caccgCGCAGGACCAGACCCCATAA-3’ (sense) and 5’-aaacTTATGGGGTCTGGTCCTGCGc-3’ (anti-sense) to clone the guide sequence into pX458 (Ran et al., 2013) sgRNA scaffold (Figure 3A). cacc (lower case) in the first oligo and aaac (lower case) in the second oligo are overhangs for cloning into BbsI-site in pX458. We also add “g” between cacc and the target sequence in the first oligo because the human U6 promoter prefers “g” at the transcription start site for strong expression. Also, you may design primers using CHOPCHOP to amplify the target site for further evaluation of sgRNAs using the T7 endonuclease I assay (see Procedure C).

  2. Cloning of sgRNAs into the spCas9 expressing plasmid (pX458)
    1. Digestion of pX458 with BbsI:
      X μl
      pX458 (10 μg)
      5 μl
      10x NEB cut smart
      1 μl
      BbsI-HF
      24 - X μl
      MilliQ water
      30 μl
      in total
    2. Incubate at 37 °C overnight.
    3. Gel purify the plasmid using QIAquick Gel extraction kit. Elute the plasmid with 50 μl of 1x Tris-EDTA buffer.
    4. Annealing of oligonucleotides encoding sgRNAs:
      1 μl
      oligonucleotide, sense (100 μM)
      1 μl
      oligonucleotide, antisense (100 μM)
      8 μl
      1x Tris-EDTA buffer
      10 μl
      in total
    5. Incubate at 95 °C for 5 min. Then cool down to room temperature by switching off the thermocycler (30 min).
    6. Prepare the ligation reaction as following:
      1 μl
      diluted (1:100 with 1x Tris-EDTA buffer) annealed oligonucleotides
      1 μl
      BbsI-digested pX458 (50 ng/μl)
      5 μl
      2x ligation buffer
      1 μl
      Quick ligase
      2 μl
      MilliQ water
      10 μl
      in total
    7. Incubate at room temperature for 10 min.
    8. Add 5 μl of the ligation reaction to 50 μl of NEB10-beta.
    9. Next place on ice for 10 min.
    10. Heat shock at 42 °C for 30 s.
    11. Transfer onto ice for 3 min.
    12. Plate the reaction on an LB-Agar plate containing ampicillin (50 μg/ml).
    13. Incubate at 37 °C overnight.
    14. Inoculate 2 ml LB broth containing ampicillin (50 μg/ml) from a single colony and incubate at 37 °C overnight with agitation.
      Note: 60-70% of colonies have a correct insertion in our experience. 
    15. Purify the plasmid from 1 ml of the overnight culture using QIAprep Spin Miniprep kit.
      Elute the plasmid with 50 μl of buffer EB and determine the concentration
    16. Verify the plasmid by sequencing with a primer (5’-gactatcatatgcttaccgt-3’).

  3. (Optional) Evaluation of sgRNA by T7 endonuclease I assay
    1. Plate NIH3T3 cells at the density of 2.5 x 104 cells (500 μl/well, DMEM/10% FBS/1% Penicillin /1% Streptomycin) onto a well of 24-well plate.
    2. Incubate in a 37 °C CO2 incubator overnight.
    3. Transfection
      1. Add 1.5 μl of FuGENE6 reagent to 50 μl of serum-free DMEM in a 1 ml plastic tube and mix by vortexing.
      2. Incubate the mixture for 5 min at room temperature.
      3. Add 0.5 μg of pX458-sgRNA or control vector to the mixture and mix by vortexing.
      4. Incubate the mixture for 30 min at room temperature.
      5. Add the mixture to each well of a 24-well plate containing cells.
      6. Incubate in a 37 °C CO2 incubator for 24 h.
      7. Replace the cell culture media with fresh one (DMEM/10% FBS/1% Penicillin /1% Streptomycin).
      8. Repeat Steps C3d-C3e.
        Note: Repeated transfection is essential for high expression of Cas9 and sgRNA to edit the target gene in NIH3T3 cells.
      9. Incubate in a 37 °C CO2 incubator for 48 h.
    4. Remove the supernatant of cells by pipetting and wash cells with 500 μl of 1x PBS.
    5. Add 100 μl of QuickExtract to each well and collect the lysate containing genomic DNA to a 1 ml plastic tube.
    6. Incubate at 65 °C for 15 min and then at 98 °C for 10 min.
    7. Centrifuge at 20,000 x g for 10 min.
    8. Collect 60 μl of the supernatant (can be stored at -20 °C).
    9. PCR to amplify 200-300 bp fragments around the CRISPR binding site. The primers can be designed by CHOPCHOP.
      6 μl
      lysate
      1.5 μl
      dNTP (the mixture of dTTP, dCTP, dGTP and dATP, each at a final concentration of 10 mM)
      12 μl
      5x buffer
      1.5 μl
      primer S (10 μM)
      1.5 μl
      primer AS (10 μM)
      1 μl
      PrimeSTAR HS DNA polymerase
      36.5 μl
      MilliQ water
      60 μl
      in total
    10. Start PCR with the following settings:
      1. Denaturation at 95 °C for 2 min.
      2. 35 cycles of
        95 °C for 10 s
        55 °C for 5 s
        72 °C for 45 s
      3. Final elongation at 72 °C for 2 min.
      4. Store at 4 °C.
    11. Gel purify PCR product using QIAquick Gel extraction kit. Elute PCR product with 50 μl of buffer EB.
      1. Denaturarion and annealing of PCR product:
        5 μl PCR product (200 ng)
        2 μl 10x NEB buffer 2
        12 μl MilliQ water
        19 μl in total
      2. 95 °C for 5 min. 
      3. Cool down to room temperature by switching off the thermocycler (30 min).
    12. Add 1 μl of diluted (1:5) T7 endonuclease I (2.5 U/μl).
      Note: Dilution of T7 endonuclease I is essential because an excess amount of the enzyme will result in non-specific cleavage of DNA.
    13. Incubate at 37 °C for 30 min.
    14. Run on 2% agarose gel and verify bands in sgRNA transfected-wells compared with control wells (Figure 2).


      Figure 2. Agarose gel electrophoresis of T7 endonuclease I-digested PCR products derived from NIH3T3 cells transfected with sgRNA and Cas9. The target exon was PCR-amplified and gel purified. After the denaturation and annealing, the PCR product was digested by T7 endonuclease I and run on 2% agarose gel. The asterisk indicates cleaved DNA fragment. In this experiment, we found only one shorter fragment. However, you may find bands of two different sizes after the cleavage if you carefully design the primers. It can be useful to design PCR primers such that the indel resulting from genome editing is not in the middle of the amplicon, ultimately resulting in two cleaved bands of different sizes.

      You can quantify the efficiency of gene modification based on the result of T7 endonuclease I assay. The percentage of cleavage on a gel could be converted to the percentage of gene modification by using an equation below.

      % gene modification = 100 x (1 - (1 - fraction cleaved)1/2 )(Guschin et al., 2010)

      Also, you may evaluate the efficiency of gene editing by a webtool called Synthego’s ICE (https://ice.synthego.com/#/). Target exons from edited or unedited (control) cells are PCR-amplified and Sanger sequenced. You may submit the results of sequencing to the webtool and ICE compares these sequence traces to give a detailed analysis of gene editing.

  4. Tandem cloning of two sgRNAs into pAAV EF1α DIO mCherry
    In Procedure A, you clone several sgRNAs targeting the same gene. For the efficient gene disruption, you tandemly clone two different sgRNAs driven by independent human U6 promoters into an AAV vector (Figures 3B and 3C) using the Gibson Assembly method.


    Figure 3. Construction strategy of a dual sgRNA viral vector. A. Cloning of guide oligonucleotides. Oligonucleotides with 20-nt guide sequence and overhangs for cloning into BbsI site in pX458 were annealed and cloned into BbsI-digested pX458. B. Cloning of U6 promoter-driven sgRNAs. U6 promoter followed by sgRNA1 or sgRNA2 were PCR amplified, respectively, by using primers with overhangs for Gibson Assembly. C. Gibson Assembly of the dual sgRNA virus vector. U6-gRNA1 and U6-gRNA2 were cloned into MluI-digested pAAV EF1 DIO mCherry using Gibson Assembly.

    1. Digestion, dephosphorylation and purification of pAAV EF1α DIO mCherry with MluI.
      1. Digestion:
        X μl
        pAAV EF1α DIO mCherry (10 μg)
        5 μl
        10x NEB cut smart
        1 μl
        MluI-HF
        24 - X μl
        MilliQ water
        30 μl
        in total
      2. Incubate at 37 °C overnight.
      3. Add 1 μl of CIP, and then incubate at 37 °C for 60 min.
      4. Gel purify the plasmid using QIAquick Gel extraction kit. 
      5. Elute the plasmid with 50 μl of buffer EB.
    2. Amplification of U6 promoter plus sgRNA#1 or sgRNA#2.
      1. PCR reaction#1 (Expected size is 393 bp):
        3 μl
        pX458-sgRNA#1 (10 ng/μl)
        1.5 μl
        dNTP, 10 mM each
        12 μl
        5x buffer
        1.5 μl
        primer S1 (10 μM); 5’- taggggttcctgcggccgcacgcgtgagggcctatttc -3’
        1.5 μl
        primer AS1 (10 μM); 5’- ataggccctctctagaaaaaaagcaccgactc -3’
        1 μl
        PrimeSTAR HS DNA polymerase
        39.5 μl
        MilliQ water
        60 μl
        in total
      2. PCR reaction#2 (Expected size is 388 bp):
        3 μl
        pX458-sgRNA#2 (10 ng/μl)
        1.5 μl
        dNTP, 10 mM
        12 μl
        5x buffer
        1.5 μl
        primer S2 (10 μM); 5’- tttttctagagagggcctatttcccatg -3’
        1.5 μl
        primer AS2 (10 μM); 5’- atccatctttgcaaagcttacgcgtaaaaaagcaccgac -3’
        1 μl
        PrimeSTAR HS DNA polymerase
        39.5 μl
        MilliQ water
        60 μl
        in total
    3. Start PCR with the following settings.
      1. Denaturation at 95 °C for 2 min.
      2. 35 cycles of
        95 °C for 10 s
        62 °C for 5 s
        72 °C for 45 s
      3. Final elongatation at 72 °C for 2 min.
      4. Store at 4 °C.
    4. Run on a 2% agarose gel.
    5. Gel purify the PCR product using QIAquick Gel extraction kit. Elute the PCR product with 50 μl of MilliQ water.
    6. Digestion of the residual template plasmid:
      45 μl
      PCR amplicon#1 or #2
      5 μl
      10x NEB cut smart
      1 μl
      DpnI
      51 μl
      in total
    7. Incubate at 37 °C for 30 min, 80 °C for 30 min.
    8. Gibson assembly:
      1 μl
      CIP-treated MluI-digested pAAV EF1α DIO mCherry (100 ng/μl)
      3 μl
      PCR amplicon#1
      3 μl
      PCR amplicon#2
      3 μl
      MilliQ water
      10 μl
      Gibson Assembly mix
      20 μl
      in total
    9. Incubate at 50 °C for 15 min.
    10. Add 2 μl of ligation reaction to 50 μl of NEB10-beta.
    11. Follow Steps B9-B15.
    12. Verify the plasmid by sequencing with a primer (e.g., 5’-actgacgggcaccggagcca-3’).
    13. Proceed to virus packaging.
      Note: We usually request virus packaging services from Stanford Neuroscience Gene Vector and Virus core.
    14. Keep the virus stock at -80 °C.
      Note: This strategy can disrupt genes in a cell type-specific manner. You can use the virus not only for adult mouse brain but also other organs.

Recipes

All materials and reagents are commercially available.

Acknowledgments

H.Y. was supported by Uehara memorial foundation research fellowship. L.d.L was supported by National Institute of Health Grants AG047671, MH087592, MH102638. This protocol was adapted from Yamaguchi et al. (2018) and includes a recent improvement of the method of our lab.

Competing interests

Competing interestsThe authors declare no competing financial interests.

References

  1. Labun, K., Montague, T. G., Gagnon, J. A., Thyme, S. B. and Valen, E. (2016). CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res 44(W1): W272-276.
  2. Platt, R. J., Chen, S., Zhou, Y., Yim, M. J., Swiech, L., Kempton, H. R., Dahlman, J. E., Parnas, O., Eisenhaure, T. M., Jovanovic, M., Graham, D. B., Jhunjhunwala, S., Heidenreich, M., Xavier, R. J., Langer, R., Anderson, D. G., Hacohen, N., Regev, A., Feng, G., Sharp, P. A. and Zhang, F. (2014). CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159(2): 440-455.
  3. Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A. and Zhang. F. (2013). Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11): 2281-2308.
  4. Swiech, L., Heidenreich, M., Banerjee, A., Habib, N., Li, Y., Trombetta, J., Sur, M. and Zhang, F. (2015). In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol 33(1): 102-106.
  5. Yamaguchi, H., Hopf, F. W., Li, S. B. and de Lecea, L. (2018). In vivo cell type-specific CRISPR knockdown of dopamine beta hydroxylase reduces locus coeruleus evoked wakefulness. Nat Commun 9(1): 5211.
  6. Guschin, D. Y., Waite, A. J., Katibah, G. E., Miller, J. C., Holmes, M. C. and Rebar, E. J. (2010). A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol 649: 247-256.

简介

最近开发的基于工程CRISPR / Cas9系统的基因编辑技术使研究人员能够在成年小鼠脑中以细胞类型特异性方式破坏基因。使用这些技术,我们最近发现蓝斑蓝斑(LC)去甲肾上腺素神经元中的多巴胺β-羟化酶基因在维持觉醒中起着至关重要的作用。我们的方法包括四个步骤,(1)将Cre依赖的spCas9敲入小鼠与Cre-驱动小鼠杂交以在目标神经群体中表达spCas9,(2)克隆sgRNA,(3)构建AAV(腺相关的)病毒)载体表达双重sgRNA,和(4)病毒包装和立体定位注射病毒进入目标脑区。在这里,我们描述了AAV载体构建的详细方案,用于成年小鼠脑中的细胞类型特异性CRISPR基因编辑。该方法采用双sgRNA策略有效破坏靶基因。首先,将靶向相同基因的几种不同sgRNA克隆到表达spCas9的质粒中。在通过T7核酸内切酶测定法评估sgRNA后,使用Gibson组装方法将两种最有效的sgRNA串联克隆到AAV载体中。
【背景】在大脑中特定细胞亚型中检测基因功能仍然是一个挑战。诸如基于RNAi的方法的常规技术缺乏细胞特异性和效率。另一方面,最近开发的使用CRISPR / Cas9系统的基因编辑技术允许研究人员以细胞类型特异性方式有效地破坏基因。由Feng Zhang领导的一个研究小组通过AAV介导的sgRNA传递显示了成年小鼠脑中多个基因的破坏(Swiech et al。,2015)。此外,该组建立了cre依赖性spCas9敲入小鼠(Platt et al。,2014)。在最近的一项研究中,我们使用携带双重sgRNA的AAV载体来提高双等位基因靶向的效率(Yamaguchi et al。,2018)。我们在这里描述了构建双重sgRNA AAV载体的方案(图1)。

图1.所有程序的示意图。我们的方法包括四个步骤:(1)将Cre依赖性spCas9敲入小鼠与Cre-驱动小鼠品系杂交以在目标神经群体中表达spCas9, (2)将sgRNA克隆到表达spCas9的质粒中,(3)构建串联表达双重sgRNA的AAV载体,和(4)将AAV包装和立体定位病毒注射到靶脑区域。

关键字:CRISPR/Cas9, AAV, 成年小鼠脑组织, Gibson 装配, 双sgRNA

材料和试剂

  1. 移液器吸头
  2. 24孔板(涂层)(Thermo Fisher Scientific,目录号:142475)
  3. 1毫升塑料管
  4. NEB10-beta主管 E。大肠杆菌(New England Biolabs,目录号:C3019)
  5. pX458(Addgene,目录号:48138)
  6. pAAVEF1αDIOmCherry(Addgene,目录号:20299)
  7. BbsI-HF(New England Biolabs,目录号:R3539)
  8. MluI-HF(New England Biolabs,目录号:R3198)
  9. DpnI(New England Biolabs,目录号:R0176)
  10. 碱性磷酸酶,小牛肠道(CIP)(New England Biolabs,目录号:M0290)
  11. 引导寡核苷酸(参见程序A)
  12. 快速结扎套件(New England Biolabs,目录号:M2200)
  13. Gibson Assembly Master Mix(新英格兰Biolabs,目录号:E2611)
  14. PrimeSTAR HS DNA聚合酶(Takara,目录号:R010)
  15. dNTP每种混合10 mM(10 mM dTTP,dCTP,dGTP和dATP的混合物)(GenScript,目录号:C01689)
  16. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
  17. QIAprep Spin Miniprep试剂盒(QIAGEN,目录号:27104)
  18. Tris-EDTA,(1x溶液)(Fisher BioReagents,目录号:BP2473)
  19. LB肉汤EZMix粉末(Sigma,目录号:L7658)
  20. LB Agar(Fisher BioReagents,目录号:BP1425)
  21. 氨苄青霉素(Gemini Bio-Products,目录号:400-130P)
  22. 验证将sgRNA克隆到pX458中的引物:5'-gactatcatatgcttaccgt-3'
  23. 验证串联克隆sgRNA到pAAV的引物:5'-actgacgggcaccggagcca-3'
  24. 吉布森大会的入门书:
    Primer S1:5'-taggggttcctgcggccgcacgcgtgagggcctatttc-3'
    Primer AS1:5'-ataggccctctctagaaaaaaagcaccgactc-3'
    Primer S2:5'-tttttctagagagggcctatttcccatg-3'
    Primer AS2:5'-atccatctttgcaaagcttacgcgtaaaaaagcaccgac-3'

    可选(参见程序C)

  25. NIH3T3细胞(ATCC,目录号:CRL-1658)
  26. AAV载体(pX458-sgRNA或pX458空作为阴性对照)
  27. 引物:您可以设计引物以通过CHOPCHOP扩增目标外显子(参见程序C)
  28. PrimeSTAR HS DNA聚合酶
  29. FuGENE6转染试剂(Promega,目录号:E2693)
  30. DMEM(1x)(Gibco,目录号:11995-065)
  31. 胎儿牛血清(康宁,目录编号:35-015-CV)
  32. 青霉素/链霉素(Gibco,目录号:15070-063)
  33. PBS,pH 7.4(Gibco,目录号:10010-023)
  34. TrypLE Select(1x)(Gibco,目录号:12563-011)
  35. QuickExtract DNA Extraction Solution(Epicenter,目录号:QE0905T)
  36. T7核酸内切酶I(New England Biolabs,目录号:M0302)

设备

  1. 移液器(Gilson,Pipetman经典P10,P20,P200)
  2. 离心机(Eppendorf,离心机5424)
  3. 孵化器(VWR,孵化5000IR轨道振动筛)
  4. 热循环仪(Applied Biosystems,GeneAmp PCR system 2700)
  5. 电泳室(Labnet,ENDURO GEL XL)
  6. (可选)CO 2 培养箱(Binder,CB 170)
  7. (可选)洁净工作台(Labconco,净化器II级生物安全柜)
  8. (可选)离心(Thermo Scientific,Soryall legend XTR)

程序

  1. sgRNA的设计
    可以通过使用gRNA设计webtools设计具有高效率和特异性的单指导RNA(sgRNA)。我们建议使用CHOPCHOP( http://chopchop.cbu.uib.no ),因为它方便用户使用界面(Labun et al。,2016)。另外,您可以在Feng Zhang实验室的网站上找到指南设计资源( https://zlab.bio/指导设计资源)。 sgRNA应靶向早期外显子(转录起始位点附近)或基因功能的基本外显子。我们通常在没有起始密码子的早期外显子上选择4到6个靶位点,因为起始密码子附近的移码突变可导致其他框内ATG的翻译,导致功能蛋白的表达。如果靶序列是5'-CGCAGGACCAGACCCCATAATGG-3'[原型间隔区相邻基序(PAM)标记为蓝色],则合成5'-caccgCGCAGGACCAGACCCCATAA-3'(有义)和5'-aaacTTATGGGGTCTGGTCCTGCGc-3'(反义)至将引导序列克隆到pX458(Ran 等人,,2013)sgRNA支架中(图3A)。第一寡核苷酸中的cacc(小写)和第二寡核苷酸中的aaac(小写)是pX458中克隆到BbsI位点的突出端。我们还在cacc和第一寡核苷酸中的靶序列之间添加“g”,因为人U6启动子在转录起始位点优选“g”用于强表达。此外,您可以使用CHOPCHOP设计引物以扩增靶位点,以使用T7核酸内切酶I测定进一步评估sgRNA(参见程序C)。

  2. 将sgRNA克隆到表达spCas9的质粒(pX458)中
    1. 用BbsI消化pX458:
      class =“ke-zeroborder”bordercolor =“#000000”style =“width:400px;” border =“0”cellspacing =“0”cellpadding =“2”>Xμl
      pX458(10μg)
      5μl
      10倍NEB削减智能
      1μl
      BbsI-HF
      24 - Xμl
      MilliQ水
      30μl
      总计
    2. 在37°C孵育过夜。
    3. 使用QIAquick凝胶提取试剂盒凝胶纯化质粒。用50μl1xTris-EDTA缓冲液洗脱质粒。
    4. 退火编码sgRNA的寡核苷酸: class =“ke-zeroborder”bordercolor =“#000000”style =“width:450px;” border =“0”cellspacing =“0”cellpadding =“2”>1μl
      寡核苷酸,有义(100μM)
      1μl
      寡核苷酸,反义(100μM)
      8μl
      1x Tris-EDTA缓冲液
      10μl
      总计
    5. 在95°C孵育5分钟。然后关闭热循环仪(30分钟)冷却至室温。
    6. 准备连接反应如下: class =“ke-zeroborder”bordercolor =“#000000”style =“width:700px;” border =“0”cellspacing =“0”cellpadding =“2”>1μl
      用1x Tris-EDTA缓冲液稀释(1:100)退火的寡核苷酸
      1μl
      BbsI消化的pX458(50 ng /μl)
      5μl
      2x结扎缓冲液
      1μl
      快速连接
      2μl
      MilliQ水
      10μl
      总计
    7. 在室温下孵育10分钟。
    8. 将5μl连接反应物加入50μlNEB10-β中。
    9. 接下来放在冰上10分钟。
    10. 在42°C下热冲击30秒。
    11. 转移到冰上3分钟。
    12. 将反应物在含有氨苄青霉素(50μg/ ml)的LB-琼脂平板上铺板。
    13. 在37°C孵育过夜。
    14. 从单个菌落接种含有氨苄青霉素(50μg/ ml)的2ml LB肉汤,并在37℃下搅拌过夜。
      注意:根据我们的经验,60-70%的菌落具有正确的插入。 
    15. 使用QIAprep Spin Miniprep试剂盒从1 ml过夜培养物中纯化质粒。
      用50μl缓冲液EB洗脱质粒并测定浓度
    16. 通过引物(5'-gactatcatatgcttaccgt-3')测序验证质粒。

  3. (可选)通过T7核酸内切酶I测定评估sgRNA
    1. 将密度为2.5×10 4 细胞(500μl/孔,DMEM / 10%FBS / 1%青霉素/ 1%链霉素)的NIH3T3细胞置于24孔板的孔中。 />
    2. 在37°C CO 2 培养箱中孵育过夜。
    3. 转染
      1. 将1.5μlFuGENE6试剂加入1ml塑料管中的50μl无血清DMEM中,并通过涡旋混合。
      2. 在室温下孵育混合物5分钟。
      3. 向混合物中加入0.5μgpX458-sgRNA或对照载体,并通过涡旋混合。
      4. 在室温下孵育混合物30分钟。
      5. 将混合物加入含有细胞的24孔板的每个孔中。
      6. 在37°C CO 2 培养箱中孵育24小时。
      7. 用新鲜培养基替换细胞培养基(DMEM / 10%FBS / 1%青霉素/ 1%链霉素)。
      8. 重复步骤C3d-C3e。
        注意:重复转染对于高表达Cas9和sgRNA以编辑NIH3T3细胞中的靶基因至关重要。
      9. 在37°C CO 2 培养箱中孵育48小时。
    4. 通过移液移除细胞上清液,并用500μl1xPBS洗涤细胞。
    5. 每孔加入100μlQuickExtract,将含有基因组DNA的裂解液收集到1 ml塑料管中。
    6. 在65°C孵育15分钟,然后在98°C孵育10分钟。
    7. 在20,000 x g 离心10分钟。
    8. 收集60μl上清液(可以在-20°C下储存)。
    9. PCR扩增CRISPR结合位点周围的200-300bp片段。引物可以通过CHOPCHOP设计。
      class =“ke-zeroborder”bordercolor =“#000000”style =“width:800px;” border =“0”cellspacing =“0”cellpadding =“2”>6μl
      裂解物
      1.5μl
      dNTP(dTTP,dCTP,dGTP和dATP的混合物,各自的终浓度为10 mM)
      12μl
      5x缓冲区
      1.5μl
      引物S(10μM)
      1.5μl
      引物AS(10μM)
      1μl
      PrimeSTAR HS DNA聚合酶
      36.5μl
      MilliQ水
      60μl
      总计
    10. 使用以下设置开始PCR:
      1. 在95℃变性2分钟。

      2. 的35个循环 95°C 10秒
        55°C,持续5秒
        72°C,45秒
      3. 在72℃下最终伸长2分钟。
      4. 储存在4°C。
    11. 使用QIAquick凝胶提取试剂盒凝胶纯化PCR产物。用50μl缓冲液EB洗脱PCR产物。
      1. PCR产物的变性和退火:
        5μlPCR产物(200 ng)
        2μl10xNEB缓冲液2
        12μlMilliQ水
        总共19μl
      2. 95°C,5分钟。 
      3. 关闭热循环仪(30分钟)冷却至室温。
    12. 加入1μl稀释的(1:5)T7核酸内切酶I(2.5U /μl)。
      注意:T7核酸内切酶I的稀释是必需的,因为过量的酶会导致DNA的非特异性切割。
    13. 在37°C孵育30分钟。
    14. 在2%琼脂糖凝胶上运行并验证sgRNA转染孔中的条带与对照孔相比(图2)。


      图2.来自用sgRNA和Cas9转染的NIH3T3细胞的T7核酸内切酶I消化的PCR产物的琼脂糖凝胶电泳。将靶外显子进行PCR扩增并凝胶纯化。变性和退火后,用T7内切核酸酶I消化PCR产物,并在2%琼脂糖凝胶上电泳。星号表示切割的DNA片段。在这个实验中,我们发现只有一个较短的片段。但是,如果仔细设计引物,您可能会在切割后发现两种不同大小的条带。设计PCR引物可能是有用的,因为基因组编辑产生的插入缺失不在扩增子的中间,最终导致两个不同大小的切割条带。

      您可以根据T7核酸内切酶I检测结果量化基因修饰的效率。通过使用下面的等式,可以将凝胶上的切割百分比转换为基因修饰的百分比。

      %基因修饰= 100 x(1 - (1 - 分裂切割) 1/2 )(Guschin et al。,2010)
      此外,您可以通过名为Synthego的ICE( https://ice.synthego.com)的webtool评估基因编辑的效率。 /#/ )。来自编辑或未编辑(对照)细胞的靶外显子进行PCR扩增和Sanger测序。您可以将测序结果提交给webtool,ICE会比较这些序列迹线,以便对基因编辑进行详细分析。

  4. 将两个sgRNA串联克隆到pAAVEF1αDIOmCherry中 在程序A中,您克隆了几个靶向相同基因的sgRNA。为了有效的基因破坏,使用Gibson组装方法将由独立的人U6启动子驱动的两种不同的sgRNA串联克隆到AAV载体中(图3B和3C)。


    图3.双重sgRNA病毒载体的构建策略。 A.引导寡核苷酸的克隆。将具有20-nt指导序列和用于克隆到pX458中的BbsI位点的突出端的寡核苷酸退火并克隆到BbsI消化的pX458中。 B.克隆U6启动子驱动的sgRNA。通过使用具有用于Gibson组装的突出端的引物,分别PCR扩增U6启动子,接着是sgRNA1或sgRNA2。 C. Gibson装配双重sgRNA病毒载体。使用Gibson Assembly将U6-gRNA1和U6-gRNA2克隆到MluI消化的pAAVEF1DIOmCherry中。

    1. 用MluI消化,去磷酸化和纯化pAAVEF1αDIOmCherry。
      1. 消化: class =“ke-zeroborder”bordercolor =“#000000”style =“width:450px;” border =“0”cellspacing =“0”cellpadding =“2”>Xμl
        pAAVEF1αDIOmCherry(10μg)
        5μl
        10倍NEB削减智能
        1μl
        MluI-HF
        24 - Xμl
        MilliQ水
        30μl
        总计
      2. 在37°C孵育过夜。
      3. 加入1μlCIP,然后在37°C孵育60分钟。
      4. 使用QIAquick凝胶提取试剂盒凝胶纯化质粒。 
      5. 用50μl缓冲液EB洗脱质粒。
    2. 扩增U6启动子加上sgRNA#1或sgRNA#2。
      1. PCR反应#1(预期大小为393 bp): class =“ke-zeroborder”bordercolor =“#000000”style =“width:650px;” border =“0”cellspacing =“0”cellpadding =“2”>3μl
        pX458-sgRNA#1(10 ng /μl)
        1.5μl
        dNTP,每种10 mM
        12μl
        5x缓冲区
        1.5μl
        引物S1(10μM); 5'-taggggttcctgcggccgcacgcgtgagggcctatttc -3'
        1.5μl
        引物AS1(10μM); 5'- ataggccctctctagaaaaaaagcaccgactc -3'
        1μl
        PrimeSTAR HS DNA聚合酶
        39.5μl
        MilliQ水
        60μl
        总计
      2. PCR反应#2(预期大小为388 bp): class =“ke-zeroborder”bordercolor =“#000000”style =“width:650px;” border =“0”cellspacing =“0”cellpadding =“2”>3μl
        pX458-sgRNA#2(10 ng /μl)
        1.5μl
        dNTP,10 mM
        12μl
        5x缓冲区
        1.5μl
        引物S2(10μM); 5'-tttttctagagagggcctatttcccatg -3'
        1.5μl
        引物AS2(10μM); 5'- atccatctttgcaaagcttacgcgtaaaaaagcaccgac -3'
        1μl
        PrimeSTAR HS DNA聚合酶
        39.5μl
        MilliQ水
        60μl
        总计
    3. 使用以下设置启动PCR。
      1. 在95℃变性2分钟。

      2. 的35个循环 95°C 10秒
        62°C,持续5秒
        72°C,45秒
      3. 最后在72℃下伸长2分钟。
      4. 储存在4°C。
    4. 用2%琼脂糖凝胶电泳。
    5. 使用QIAquick凝胶提取试剂盒凝胶纯化PCR产物。用50μlMilliQ水洗脱PCR产物。
    6. 消化残留的模板质粒:
      class =“ke-zeroborder”bordercolor =“#000000”style =“width:350px;” border =“0”cellspacing =“0”cellpadding =“2”>45μl
      PCR扩增子#1或#2
      5μl
      10倍NEB削减智能
      1μl
      DpnI
      51μl
      总计
    7. 在37°C孵育30分钟,在80°C孵育30分钟。
    8. 吉布森大会: class =“ke-zeroborder”bordercolor =“#000000”style =“width:650px;” border =“0”cellspacing =“0”cellpadding =“2”>1μl
      经CIP处理的MluI消化的pAAVEF1αDIOmCherry(100 ng /μl)
      3μl
      PCR扩增子#1
      3μl
      PCR扩增子#2
      3μl
      MilliQ水
      10μl
      吉布森大会组合
      20μl
      总计
    9. 在50°C孵育15分钟。
    10. 向50μlNEB10-β中加入2μl连接反应物。
    11. 按照步骤B9-B15。
    12. 通过用引物(例如,5'-actgacgggcaccggagcca-3')测序验证质粒。
    13. 继续进行病毒包装。
      注意:我们通常从 斯坦福神经科学基因载体和病毒核心 。
    14. 将病毒储存在-80°C。
      注意:此策略可以以特定于细胞类型的方式破坏基因。您不仅可以将病毒用于成年小鼠大脑,还可以用于其他器官。

食谱

所有材料和试剂均可商购。

致谢

H.Y.得到了上原纪念基金研究奖学金的支持。 L.d.L得到国立卫生研究院资助AG047671,MH087592,MH102638的支持。该协议改编自Yamaguchi et al。(2018),并包括我们实验室方法的最新改进。

利益争夺

竞争利益作者宣称没有竞争性的经济利益。

参考

  1. Labun,K.,Montague,T.G。,Gagnon,J.A.,Thyme,S.B。和Valen,E。(2016)。 CHOPCHOP v2:下一代CRISPR基因组工程的网络工具。 核酸Res 44(W1):W272-276。
  2. Platt,RJ,Chen,S.,Zhou,Y.,Yim,MJ,Swiech,L.,Kempton,HR,Dahlman,JE,Parnas,O.,Eisenhaure,TM,Jovanovic,M.,Graham,DB,Jhunjhunwala ,S.,Heidenreich,M.,Xavier,RJ,Langer,R.,Anderson,DG,Hacohen,N.,Regev,A.,Feng,G.,Sharp,PA和Zhang,F。(2014)。 CRISPR-Cas9敲入小鼠进行基因组编辑和癌症建模。 细胞 159(2):440-455。
  3. Ran,F.A.,Hsu,P。D.,Wright,J.,Agarwala,V.,Scott,D。A.和Zhang。 F.(2013)。 使用CRISPR-Cas9系统的基因组工程。 Nat Protoc 8(11):2281-2308。
  4. Swiech,L.,Heidenreich,M.,Banerjee,A.,Habib,N.,Li,Y.,Trombetta,J.,Sur,M。和Zhang,F。(2015)。使用CRISPR-Cas9 体内在哺乳动物大脑中查询基因功能。 Nat Biotechnol 33(1):102-106。
  5. Yamaguchi,H.,Hopf,F.W.,Li,S.B。和de Lecea,L。(2018)。 体内细胞类型特异性CRISPR敲低多巴胺β羟化酶可减少基因座coeruleus唤起了觉醒。 Nat Commun 9(1):5211。
  6. Guschin,D.Y.,Waite,A.J.,Katibah,G.E.,Miller,J.C.,Holmes,M.C。和Rebar,E.J。(2010)。 监测内源基因修饰的快速通用检测方法。 方法Mol Biol 649:247-256。
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yamaguchi, H. and de Lecea, L. (2019). Construction of Viral Vectors for Cell Type-specific CRISPR Gene Editing in the Adult Mouse Brain. Bio-protocol 9(16): e3334. DOI: 10.21769/BioProtoc.3334.
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