CRISPR-PCS Protocol for Chromosome Splitting and Splitting Event Detection in Saccharomyces cerevisiae
检测酿酒酵母中染色体分裂和分裂事件的CRISPR-PCS实验方案   

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Scientific Reports
Aug 2016

 

Abstract

Chromosome engineering is an important technology with applications in basic biology and biotechnology. Chromosome splitting technology called PCS (PCR-mediated Chromosome Splitting) has already been developed as a fundamental chromosome engineering technology in the budding yeast. However, the splitting efficiency of PCS technology is not high enough to achieve multiple splitting at a time. This protocol describes a procedure for achieving simultaneous and multiple chromosome splits in the budding yeast Saccharomyces cerevisiae by a new technology called CRISPR-PCS. At least four independent sites in the genome can be split by one transformation. Total time and labor for obtaining a multiple split yeast strain is drastically reduced when compared with conventional PCS technology.

Keywords: Saccharomyces cerevisiae (酿酒酵母), Chromosome engineering (染色体工程), CRISPR/Cas9 (CRISPR/Cas9), Chromosome splitting (染色体分裂)

Background

Chromosome engineering technologies that enable rapid and efficient manipulation of multiple genetic loci or chromosomal regions have become increasingly important. Such technologies offer a powerful means for elucidating chromosome and genome function. Additionally, it can be used for breeding useful strains through the creation of a wide array of genetic variants. A chromosome splitting technology called PCS (PCR-mediated Chromosome Splitting) technology has been developed in the budding yeast Saccharomyces cerevisiae. This technology allows the splitting of yeast chromosomes at any desired site by introducing centromeres and telomere seed sequences based on the homologous recombination mechanism. The resulting chromosomes possess one centromere and telomeres at both ends, thus function as normal chromosomes (Sugiyama et al., 2005). However, low splitting efficiency is a drawback in PCS, therefore simultaneous and multiple splitting of chromosomes has been impossible. In this situation, we developed a novel chromosome splitting technology called CRISPR-PCS. It is well known that double strand break (DSB) markedly increases homologous recombination activity around the DSB site in yeast (Agmon et al., 2009). The CRISPR/Cas9 system is a genome editing technology that can induce targeted DSBs. By utilizing CRISPR/Cas9 system, we can induce DSB at any genomic locus and thus activate homologous recombination activity. CRISPR-PCS is a technology that combines CRISPR/Cas9 system with PCS, thus allowing the increase of splitting efficiency by approximately 200 fold. This drastically increased efficiency enables simultaneous and multiple chromosome spitting. Overview of the CRISPR-PCS technology is illustrated in Figure 1.


Figure 1. Overview of CRISPR-PCS. In CRISPR-PCS, one gRNA expressing plasmid for the specific targeting site and two splitting modules are required to split yeast chromosome at a specific site. These DNA molecules are introduced into the Cas9 expressing strain, i.e., the strain carrying p414-TEF1p-Cas9-CYC1t plasmid. Transformants where the expected split event occurred are selected by auxotrophic marker selection. Closed black circles represent the centromere. Red and blue boxes represent the homology sequences for recombination. Arrows represent the telomere sequence.

Materials and Reagents

  1. 10-100 μl pipette tips (e.g., Greiner Bio One International, catalog number: 685280 )
  2. 100-1,000 μl pipette tips (e.g., Greiner Bio One International, catalog number: 686290 )
  3. PCR tube (e.g., Greiner Bio One International, catalog number: 683201 )
  4. p426-SNR52p-gRNA.CAN1.Y-SUP4t (Addgene, catalog number: 43803 )
  5. p414-TEF1p-Cas9-CYC1t (Addgene, catalog number: 43802 )
  6. Escherichia coli DH5α competent cell (NIPPON GENE, catalog number: 316-06233 )
  7. DNA, MB-grade from fish sperm (Roche Diagnostics, catalog number: 11467140001 )
  8. KOD plus neo (TOYOBO, catalog number: KOD-401 )
  9. 2 mM dNTP solution (Attached in the KOD plus neo)
  10. 25 mM magnesium sulfate (MgSO4) (Attached in the KOD plus neo)
  11. Oligonucleotide primer 1 for construction of a gRNA expressing plasmid (5’-N20GTTTTAGAGCTAGAAATAGCAAG-3’) (synthesized in Sigma-Aldrich Japan)
  12. Oligonucleotide primer 2 for construction of gRNA expressing plasmid (5’-cN20GATCATTTATCTTTCACTGCGGA-3’) (synthesized in Sigma-Aldrich Japan)
  13. DpnI (Takara Bio, catalog number: 1235A )
  14. QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704 )
  15. 10x NEB buffer 2 (New England Biolabs, catalog number: B7002S )
  16. BSA solution (attached in the T4 DNA polymerase) (New England Biolabs, catalog number: M0203 )
  17. 10x T4 DNA ligase buffer (New England Biolabs, catalog number: M0202 )
  18. T4 DNA polymerase (New England Biolabs, catalog number: M0203 )
  19. 2.5 mM each dNTP mix (Takara Bio, catalog number: 4030 )
  20. Ampicillin (Wako Pure Chemical Industries, catalog number: 015-10382 )
  21. Oligonucleotide primer 1 for construction of a splitting module (5’-N50GGCCGCCAGCTGAAGCTTCG-3’) (synthesized in Sigma-Aldrich Japan)
  22. Oligonucleotide primer 2 for construction of a splitting module (5’-CCCCAACCCCAACCCCAACCCCAACCCCAACCCCAAAGGCCACTAGTGGATCTGAT-3’) (synthesized in Sigma-Aldrich Japan)
  23. Oligonucleotide primer for sequencing (5’-ACGCCAAGCGCGCAATTAAC-3’) (synthesized in Sigma-Aldrich Japan)
  24. Lithium acetate dihydrate (Wako Pure Chemical Industries, catalog number: 120-01535 )
  25. Polyethylene glycol 4,000 (Wako Pure Chemical Industries, catalog number: 162-09115 )
  26. ECL Direct Nucleic Acid Labelling and Detection System (GE Healthcare, catalog number: RPN3000 )
  27. Ethidium bromide solution (10 mg/ml) (Nacalai Tesque, catalog number: 14631-94 )
  28. LB broth (Sigma-Aldrich, catalog number: L3022-1KG )
  29. Agar (Wako Pure Chemical Industries, catalog number: 010-08725 )
  30. Glucose (Wako Pure Chemical Industries, catalog number: 043-31163 )
  31. Yeast nitrogen base without amino acids (e.g., BD, Difco, catalog number: 291940 )
  32. Sodium hydroxide (NaOH) (e.g., Wako Pure Chemical Industries, catalog number: 192-15985 )
  33. Peptone (e.g., BD, BactoTM, catalog number: 211677 )
  34. Yeast extract (e.g., BD, BactoTM, catalog number: 288620 )
  35. Hydrochloric acid (HCl) (e.g., Wako Pure Chemical Industries, catalog number: 087-10361 )
  36. LB plate (see Recipes)
  37. Yeast minimum medium (SD medium) (see Recipes)
  38. YPD medium (see Recipes)

Equipment

  1. Pipettes
  2. Thermal cycler (e.g., Takara Bio, model: Dice® Touch, catalog number: TP350 ) (Use at Procedure B and Procedure C)
  3. Incubator (e.g., Panasonic Healthcare, model: MIR-H163 ) (Use at Procedure B and Procedure D)
  4. Air shaker (e.g., TAITEC, model: BR-21UM MR ) (Use at Procedure B and Procedure D)
  5. Heat block (e.g., TAITEC, model: DTU-1BN ) (Use at Procedure B, Procedure D and Procedure E)
  6. DNA sequencer (e.g., Applied Biosystems, model: ABI PRISM® 3100 Genetic Analyzer ) (Use at Procedure B)
  7. CHEF-DR® III pulsed field gel electrophoresis system (Bio-Rad Laboratories, model: CHEF-DR III Chiller System , catalog number: 1703700)

Procedure

  1. Designing of gRNA target site
    Once you decide a genomic locus to be split, search PAM sequence for Cas9 cleavage (5’-NGG-3’ or 5’-CCN-3’ for opposite strand) in the vicinity of the target locus. Decide the most appropriate 20 bp guide RNA (gRNA) target sequence so that the gRNA target sequence is not present in the DNA splitting module (see Procedure C) and the gRNA target sequence is not predicted to induce any off-target cleavages. CRISPRdirect (https://crispr.dbcls.jp/) may be helpful for designing target sequences. Paste your target sequence into the box and choose S. cerevisiae S288C genome for the specificity check. Then you can easily know the most appropriate guide RNA target sequence. It is known that the cleavage point by the CRISRP/Cas9 system is 3 bp upstream of PAM sequence (Jinek et al., 2012). It is desirable that the point to be split is designed at the same point cleaved by CRISPR/Cas9. However, this is not absolute requirement unless splitting modules don’t contain gRNA target sites.

  2. Construction of a plasmid expressing gRNA with designed 20 bp target sequence
    We used p426-SNR52p-gRNA.CAN1.Y-SUP4t as a template plasmid to construct a desired gRNA expressing plasmid by PCR. You can use any plasmids to express your desired gRNA. The following protocol is a version when p426-SNR52p-gRNA.CAN1.Y-SUP4t is used as a template plasmid. The principle for plasmid construction is based on SLIC technology (Li and Elledge, 2012).
    1. Perform PCR reaction


    2. After PCR reaction, add 1 μl of DpnI directly to the PCR tube to destroy methylated template plasmid. Incubate at 37 °C for 1 h.
    3. Perform gel electrophoresis and purify the PCR product by QIAquick Gel Extraction Kit.
    4. Purified fragment is dissolved into 50 μl of elution buffer.
    5. T4 DNA polymerase reaction to generate 5’ overhangs by 3’ exonuclease activity.


      Incubate at 22 °C for 30 min. Then add 2 μl of dNTP mix (2.5 mM each) to terminate the reaction. Keep on ice.
    6. Annealing reaction
      To 9 μl solution from step B5, add 1 μl of 10x T4 ligase buffer (NEB). Incubate at 37 °C for 30 min.
    7. Transformation of Escherichia coli
      Transform E. coli DH5α strain by introducing 2 μl of solution from step B6. After incubating at 37 °C for 1 h, spread onto LB plate containing ampicillin.
    8. Confirmation of plasmid construction by sequencing analysis
      Note: Although most of the E. coli transformants harbor the desired gRNA plasmid, it is better to check by sequencing analysis using the primer 5’-ACGCCAAGCGCGCAATTAAC-3’.

  3. Construction of a splitting module with 50 bp homology sequence
    1. After deciding a point to be split at Procedure A, design and purchase oligonucleotide primers to amplify any genetic marker gene flanked with 50 bp homology sequence corresponding to the upstream and downstream of the target splitting point at both primers (see Figure 2). For PCR templates we used a pUG6 based plasmid set (Guldener et al., 1996) for PCR amplification but with different genetic marker genes (Candida glabrata LEU2 gene, Candida glabrata HIS3 gene, URA3, and KanMX) or centromere (CEN4) (Sugiyama et al., 2005). Select appropriate plasmid as a template for PCR so that newly generated chromosomes contain one centromere. These plasmids are available at NBRP (http://yeast.lab.nig.ac.jp/nig.v2.1/index.html).


      Figure 2. Detailed illustration for construction of splitting modules. There are five template plasmids to construct splitting modules. CEN4 is a centromere sequence of the chromosome IV in S. cerevisiae. All sequences of the plasmids are identical except for those of genetic marker gene, because all template plasmids have the same plasmid backbone (pUG6 plasmid). Therefore, primer sequences required for annealing to the template plasmid is common when you use any plasmids as a template. In oligonucleotide primer 1 and 2, in addition to the sequence for annealing, 50 bp sequence that is identical to the upstream and downstream sequence of the target splitting point in the chromosome should be added, respectively. Closed black circles represent the centromere. Red and blue boxes represent a 50 bp sequence upstream and downstream from the target splitting point in the chromosome, respectively. Arrows represent the telomere sequence.

    2. PCR reaction


      After PCR reaction, purify the PCR product by gel extraction by QIAquick Gel Extraction Kit.
      Dissolve the purified splitting module DNA into 50 μl of elution buffer.

  4. Transformation
    1. Prepare a yeast strain in advance which constitutively express codon optimized Cas9 for yeast expression by introduction of p414-TEF1p-Cas9-CYC1t.
      Note: Yeast strains which can perform many auxotrophic marker selections are desirable. We use FY834 strain (MATα ura3-52 his3Δ200 leu2Δ1 lys2Δ202 trp1Δ63). FY834 strain is transformed by introduction of p414-TEF1p-Cas9-CYC1t plasmid by LiAc/PEG method. Because p414-TEF1p-Cas9-CYC1t plasmid has a TRP1 marker, transformants that can grow in the medium without tryptophan were selected.
    2. Cultivate the strain overnight in liquid yeast minimum medium (SD medium supplemented by necessary amino acids and nucleic acid base).
    3. Inoculate fresh 50 ml YPD medium with the yeast cell pre-culture to obtain an initial OD600 of approximately 0.2-0.3. Then incubate with shaking speed 140 rpm at 30 °C until OD600 reaches 0.8-1.0 (about 4 to 6 h).
    4. Mix the splitting DNA module(s) (1-5 μg each) and gRNA expressing plasmid(s) (7.5 μg each) and perform transformation by the conventional LiAc/PEG method (Gietz and Schiestl, 2007). LiAc/PEG method uses DNA MB-grade from salmon sperm, lithium acetate and polyethylene glycol 4000 (listed in the Materials and Reagents section).
      Note: In our protocol, the maximum transformation efficiency is achieved when the amount of gRNA expressing plasmid is 7.5 μg. However, multiple split transformants can be obtained even when using less gRNA expressing plasmid (for example, 1 μg).
    5. After transformation, cells are suspended in 200 μl of sterilized water and spread onto an appropriate selection plate and incubate at 30 °C for 2 or 3 days.
      Note: Selection of transformants by a genetic marker gene on a gRNA expressing plasmid is not necessary and may decrease transformation efficiency. It usually causes decreased number of transformants probably due to continual targeted Cas9-endonuclease activity.

  5. Confirmation of the splitting event by Pulsed Field Gel Electrophoresis and subsequent Southern blotting
    1. Prepare plugs in accordance with the kit protocol (e.g., CHEF Genomic DNA Plug Kits Instruction Manual, Bio-Rad Laboratories).
    2. Perform Pulsed Field Gel Electrophoresis (PFGE) with an appropriate apparatus (e.g., CHEF system in Bio-Rad Laboratories). A typical setting for PFGE is described in Sugiyama et al., 2005.
    3. After electrophoresis, stain the gel by ethidium bromide solution (1 μg/ml) to check the karyotype of the transformants.
    4. Perform membrane blotting by the conventional blotting method (Southern, 2006).
    5. Prepare probes for detection of a specific region by an appropriate DNA labeling kit (e.g., ECL Direct Nucleic Acid Labelling and Detection System) and perform probe hybridization.
      Note: A probe is a labeled single strand DNA having a specific sequence to detect the presence of DNA molecule having the same sequence with the probe. Typically, the size of a probe is from 500 bp to 1,000 bp. Although any region in the target chromosome can be used as a probe to check the splitting event, you may choose a region extending over the splitting point. When you use such a probe, two bands will appear corresponding to two chromosomes yielded by the splitting event. On the other hand if a splitting event did not occur, only one band corresponding to the intact chromosome will appear. A typical result is illustrated in Figure 3.
    6. Detect the labeled probe in accordance with the DNA labeling kit manual you use.


      Figure 3. A typical result of chromosome splitting by CRISPR-PCS. Chr. IV was split at the position 999,122.5 by CRISPR-PCS. The probe was designed to extend over the splitting point (from 998,717 to 999,302). The wild type strain (designated as WT) shows single band corresponding to intact Chr. IV, while transformants (designated as 1, 2, and 3) show two bands demonstrating that Chr. IV was split at the targeted point.

Data analysis

The number of transformants varies in every experiment. Generally, when targeting only one genomic site, several hundred or more transformants can be obtained and almost all the transformants harbor expected splitting. Even when targeting multiple sites, you can obtain several hundred transformants, although the frequency of transformants with expected splitting is decreased. Examination of transformants should yield at least one correctly split transformant out of 10 transformants.

Recipes

  1. LB plate
    2% LB broth (Sigma-Aldrich)
    1.5% agar
  2. Yeast minimum medium (SD medium)
    2% glucose
    0.67% yeast nitrogen base without amino acids (e.g., BD Difco)
    Note: If required, drop out mix containing all amino acids lacking appropriate amino acids used for auxotrophic selection can be used.
    Adjust to pH = 6.0 with NaOH
    For plate media, 2% agar is added
  3. YPD medium
    2% glucose
    2% peptone (e.g., BD BactoTM)
    1% yeast extract (e.g., BD BactoTM)
    Adjust to pH = 6.0 with HCl

Acknowledgments

This work was supported by Grant-in-Aid for Scientific Research (B) [15H04475] (to S. H.), Grant-in-Aid for Challenging Exploratory Research [26660066] (to S. H.), and Grant-in-Aid for Young Scientists (B) [15K18672] (to Y. S.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

References

  1. Agmon, N., Pur, S., Liefshitz, B. and Kupiec, M. (2009). Analysis of repair mechanism choice during homologous recombination. Nucleic Acids Res 37(15): 5081-5092.
  2. Gietz, R. D. and Schiestl, R. H. (2007). High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1): 31-34.
  3. Guldener, U., Heck, S., Fielder, T., Beinhauer, J. and Hegemann, J. H. (1996). A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24(13): 2519-2524.
  4. 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.
  5. Li, M. Z. and Elledge, S. J. (2012). SLIC: a method for sequence- and ligation-independent cloning. Methods Mol Biol 852: 51-59.
  6. Southern, E. (2006). Southern blotting. Nat Protoc 1: 518-525.
  7. Sugiyama, M., Ikushima, S., Nakazawa, T., Kaneko, Y. and Harashima, S. (2005). PCR-mediated repeated chromosome splitting in Saccharomyces cerevisiae. Biotechniques 38(6): 909-914.

简介

染色体工程是应用于基础生物学和生物技术的重要技术。染色体分裂技术称为PCS(PCR介导的染色体分裂)已经被开发为发芽酵母中的基本染色体工程技术。然而,PCS技术的分割效率不够高,不能一次实现多次分割。该协议描述了通过称为CRISPR-PCS的新技术在芽状酵母酿酒酵母中实现同时和多个染色体分裂的过程。基因组中至少四个独立的位点可以通过一次转化来分裂。与常规PCS技术相比,获得多重分裂酵母菌株的总时间和劳动力大大降低。

背景 能够快速有效地操纵多个遗传基因座或染色体区域的染色体工程技术变得越来越重要。这些技术为阐明染色体和基因组功能提供了有力的手段。此外,它可以用于通过创建广泛的遗传变体来繁殖有用的菌株。在芽殖酵母酿酒酵母(Saccharomyces cerevisiae)中已经开发了称为PCS(PCR-介导的染色体分裂)技术的染色体分裂技术。该技术允许通过基于同源重组机制引入着丝粒和端粒种子序列在任何所需位点分裂酵母染色体。所得到的染色体在两端具有一个着丝粒和端粒,因此起到正常染色体的作用(Sugiyama等人,2005)。然而,低分裂效率是PCS的缺点,因此染色体的同时和多次分裂是不可能的。在这种情况下,我们开发了一种称为CRISPR-PCS的新型染色体分裂技术。众所周知,双链断裂(DSB)显着增加酵母中DSB位点周围的同源重组活性(Agmon等人,2009)。 CRISPR / Cas9系统是可以诱导目标DSB的基因组编辑技术。通过使用CRISPR / Cas9系统,我们可以在任何基因组位点诱导DSB,从而激活同源重组活性。 CRISPR-PCS是将CRISPR / Cas9系统与PCS相结合的技术,从而可以将分离效率提高约200倍。这种大幅提高的效率使同时和多个染色体吐痰。 CRISPR-PCS技术概述如图1所示。


图1. CRISPR-PCS的概述在CRISPR-PCS中,需要将特定靶位点的一个gRNA表达质粒和两个分裂模块在特定位点分离酵母染色体。将这些DNA分子引入表达Cas9的菌株,即携带p414-TEF1p-Cas9-CYC1t质粒的菌株即。通过营养缺陷型标记选择选择发生预期分裂事件的转化体。闭合的黑色圆圈代表着丝粒。红色和蓝色框表示重组的同源性序列。箭头代表端粒序列。

关键字:酿酒酵母, 染色体工程, CRISPR/Cas9, 染色体分裂

材料和试剂

  1. 10-100μl移液器吸头(例如,Greiner Bio One International,目录号:685280)
  2. 100-1,000微升移液管吸头(例如,Greiner Bio One International,目录号:686290)
  3. PCR管(例如,Greiner Bio One International,目录号:683201)
  4. p426-SNR52p-gRNA.CAN1.Y-SUP4t(Addgene,catalog number:43803)
  5. p414-TEF1p-Cas9-CYC1t(Addgene,目录号:43802)
  6. 大肠杆菌DH5α感受态细胞(NIPPON GENE,目录号:316-06233)
  7. DNA,来自鱼类精子的MB级(Roche Diagnostics,目录号:11467140001)
  8. KOD plus neo(TOYOBO,目录号:KOD-401)
  9. 2mM dNTP溶液(加入KOD加neo)
  10. 25mM硫酸镁(MgSO 4)(附在KOD plus neo中)
  11. 用于构建表达gRNA的质粒(5'-N20GTTTTAGAGCTAGAAATAGCAAG-3')(在Sigma-Aldrich Japan中合成)的寡核苷酸引物1)
  12. 用于构建表达gRNA的质粒(5'-cN20GATCATTTATCTTTCACTGCGGA-3')的寡核苷酸引物2(在Sigma-Aldrich Japan中合成)
  13. Ip(Takara Bio,目录号:1235A)
  14. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
  15. 10x NEB缓冲液2(New England Biolabs,目录号:B7002S)
  16. BSA溶液(连接在T4 DNA聚合酶中)(New England Biolabs,目录号:M0203)
  17. 10x T4 DNA连接酶缓冲液(New England Biolabs,目录号:M0202)
  18. T4 DNA聚合酶(New England Biolabs,目录号:M0203)
  19. 2.5mM各dNTP混合物(Takara Bio,目录号:4030)
  20. 氨苄青霉素(和光纯药工业公司,目录号:015-10382)
  21. 用于构建分裂模块的寡核苷酸引物1(5'-N 50 GGCCGCCAGCTGAAGCTTCG-3')(在Sigma-Aldrich Japan中合成)
  22. 用于构建分裂模块的寡核苷酸引物2(5'-CCCCAACCCCAACCCCAACCCCAACCCCAACCCCAAAGGCCACTAGTGGATCTGAT-3')(在Sigma-Aldrich Japan中合成)
  23. 用于测序的寡核苷酸引物(5'-ACGCCAAGCGCGCAATTAAC-3')(在Sigma-Aldrich Japan中合成)
  24. 乙酸锂二水合物(Wako Pure Chemical Industries,目录号:120-01535)
  25. 聚乙二醇4000(Wako Pure Chemical Industries,目录号:162-09115)
  26. ECL直接核酸标记和检测系统(GE Healthcare,目录号:RPN3000)
  27. 溴化乙啶溶液(10mg/ml)(Nacalai Tesque,目录号:14631-94)
  28. LB肉汤(Sigma-Aldrich,目录号:L3022-1KG)
  29. 琼脂(Wako Pure Chemical Industries,目录号:010-08725)
  30. 葡萄糖(和光纯药,目录号:043-31163)
  31. 不含氨基酸的酵母氮碱(例如,BD,Difco,目录号:291940)
  32. 氢氧化钠(NaOH)(例如和光纯药工业公司,目录号:192-15985)
  33. 蛋白胨(例如,BD,Bacto TM,目录号:211677)
  34. 酵母提取物(例如,BD,Bacto TM,目录号:288620)
  35. 盐酸(HCl)(例如和光纯药,目录号:087-10361)
  36. LB板(参见食谱)
  37. 酵母最低培养基(SD培养基)(见食谱)
  38. YPD培养基(见食谱)

设备

  1. 移液器
  2. 热循环仪(例如,,Takara Bio,型号:Dice Touch ,目录号:TP350)(在程序B和程序C中使用)
  3. 孵化器(例如,松下医疗保健,型号:MIR-H163)(在程序B和程序D中使用)
  4. 空气振荡器(例如,TAITEC,型号:BR-21UM MR)(在程序B和程序D中使用)
  5. 热块(例如,TAITEC,型号:DTU-1BN)(在程序B中使用,程序D和程序E)
  6. DNA测序仪(例如,应用生物系统公司,Applied Biosystems,型号:ABI PRISM 3100 Genetic Analyzer)(在步骤B中使用)
  7. CHEF-DR ®III脉冲场凝胶电泳系统(Bio-Rad Laboratories,型号:CHEF-DR III冷却器系统,目录号:1703700)

程序

  1. 设计gRNA靶位点
    一旦您决定要分裂的基因组位点,搜索目标基因座附近的Cas9切割(5'-NGG-3'或5'-CCN-3')的PAM序列。确定最合适的20 bp指导RNA(gRNA)靶序列,使得gRNA靶序列不存在于DNA分裂模块中(参见方法C),并且不预测gRNA靶序列诱导任何脱靶裂解。 CRISPRdirect( https://crispr.dbcls.jp/)可能有助于设计目标序列。将您的目标序列粘贴到框中,然后选择。啤酒酵母S288C基因组进行特异性检测。那么你可以很容易地知道最适合的RNA靶序列。已知CRISRP/Cas9系统的切割点是PAM序列上游3 bp(Jinek等人,2012)。希望将分割点设计在与CRISPR/Cas9切割的同一点上。但是,除非分裂模块不包含gRNA靶位点,否则这不是绝对要求。

  2. 用设计的20bp靶序列构建表达gRNA的质粒
    我们使用p426-SNR52p-gRNA.CAN1.Y-SUP4t作为模板质粒,通过PCR构建所需的表达gRNA的质粒。您可以使用任何质粒来表达您想要的gRNA。以下协议是使用p426-SNR52p-gRNA.CAN1.Y-SUP4t作为模板质粒的版本。质粒构建的原理是基于SLIC技术(Li和Elledge,2012)。
    1. 进行PCR反应


    2. PCR反应后,直接向PCR管中加入1μl的DpnⅠ以破坏甲基化模板质粒。在37℃孵育1小时。
    3. 进行凝胶电泳,并通过QIAquick凝胶提取试剂盒纯化PCR产物
    4. 将纯化的片段溶解于50μl洗脱缓冲液中
    5. T4 DNA聚合酶反应,通过3'外切核酸酶活性产生5'突出端

      在22℃孵育30分钟。然后加入2μldNTP混合物(各2.5mM)终止反应。保持在冰上
    6. 退火反应
      向步骤B5的9μl溶液中加入1μl10×T4连接酶缓冲液(NEB)。在37°C孵育30分钟。
    7. 转化大肠杆菌
      变换E。通过引入2μl来自步骤B6的溶液的DH5α菌株。在37℃下孵育1小时后,铺在含有氨苄青霉素的LB平板上。
    8. 通过测序分析确认质粒构建
      注意:虽然大多数大肠杆菌转化体含有所需的gRNA质粒,但最好使用引物5'-ACGCCAAGCGCGCAATTAAC-3'进行测序分析来检查。

  3. 构建具有50bp同源序列的分裂模块
    1. 在确定在步骤A分裂点后,设计和购买寡核苷酸引物以扩增任何遗传标记基因,其侧翼具有对应于两个引物的靶分裂点上游和下游的50bp同源性序列(参见图2)。对于PCR模板,我们使用基于pUG6的质粒组(Guldener等人,1996),用于PCR扩增,但是使用不同的遗传标记基因( Candida glabrata LEU2 基因,假丝酵母HIS3基因,URA3和KanMX )或着丝粒(CEN4 )(Sugiyama等人 >。,2005)。选择合适的质粒作为PCR的模板,使新生成的染色体含有一个着丝粒。这些质粒可用于NBRP( http ://yeast.lab.nig.ac.jp/nig.v2.1/index.html )。


      图2.分割模块构建的详细说明有五个模板质粒构建分裂模块。 CEN4 是S染色体IV的着丝粒序列。酵母。由于所有模板质粒都具有相同的质粒骨架(pUG6质粒),所以质粒的所有序列除遗传标记基因之外都是相同的。因此,当您使用任何质粒作为模板时,退火到模板质粒所需的引物序列很常见。在寡核苷酸引物1和2中,除了退火序列之外,还应分别添加与染色体中目标分裂点的上游和下游序列相同的50bp序列。闭合的黑色圆圈代表着丝粒。红色和蓝色框分别表示染色体中目标分裂点上游和下游的50bp序列。箭头代表端粒序列。

    2. PCR反应


      PCR反应后,通过QIAquick Gel Extraction Kit凝胶提取纯化PCR产物 将纯化的分离模块DNA溶解于50μl洗脱缓冲液中
  4. 转型
    1. 通过引入p414-TEF1p-Cas9-CYC1t,预先制备酵母菌株,其组成型表达密码子优化的Cas9用于酵母表达。
      注意:可以进行许多营养缺陷型标记选择的酵母菌株是可取的。我们使用FY834菌株(MATαura3-52his3Δ200leu2Δ1lys2Δ202trp1Δ63)。通过LiAc/PEG法引入p414-TEF1p-Cas9-CYC1t质粒来转化FY834菌株。因为p414-TEF1p-Cas9-CYC1t质粒具有TRP1标记,所以可以在不含色氨酸的培养基中生长的转化体。
    2. 在液体酵母最低培养基(需要氨基酸和核酸碱补充的SD培养基)中培养菌株过夜。
    3. 用酵母细胞预培养物接种新鲜的50ml YPD培养基,以获得约0.2-0.3的初始OD 600。然后在30℃下以140rpm的振荡速度孵育,直到OD 600达到0.8-1.0(约4至6小时)。
    4. 将分离的DNA模块(各自为1-5μg)和表达gRNA的质粒(各7.5μg)混合,并通过常规的LiAc/PEG方法进行转化(Gietz和Schiestl,2007)。 LiAc/PEG方法使用鲑鱼精子DNA乙酸钙,乙酸锂和聚乙二醇4000(在材料和试剂部分列出)。
      注意:在我们的方案中,当表达gRNA的质粒的量为7.5μg时,达到最大的转化效率。然而,即使使用较少的表达gRNA的质粒(例如1μg),也可以获得多个分裂转化体。
    5. 转化后,将细胞悬浮于200μl灭菌水中,并铺在适当的选择板上,并在30℃下孵育2或3天。
      注意:通过gRNA表达质粒上的遗传标记基因选择转化体是不必要的并且可能降低转化效率。通常导致转化体数量减少,这可能是由于持续的目标Cas9-核酸内切酶活性
  5. 通过脉冲场凝胶电泳和随后的Southern印迹确认分裂事件
    1. 根据试剂盒协议(例如,CHEF Genomic DNA Plug Kits使用手册,Bio-Rad Laboratories)准备插头。
    2. 用适当的装置(例如Bio-Rad Laboratories的CHEF系统)进行脉冲场凝胶电泳(PFGE)。在Sugiyama等人,2005中描述了PFGE的典型设置。
    3. 电泳后,用溴化乙锭溶液(1μg/ml)对凝胶进行染色以检查转化体的核型。
    4. 通过常规的印迹法进行膜印迹(Southern,2006)
    5. 通过适当的DNA标记试剂盒(例如ECL直接核酸标记和检测系统)准备探针以检测特定区域,并进行探针杂交。
      注意:探针是具有特异性序列的标记单链DNA,以检测与探针具有相同序列的DNA分子的存在。通常,探针的大小为500bp至1,000bp。虽然目标染色体中的任何区域都可以用作检测分离事件的探针,但您可以选择在分割点上延伸的区域。当您使用这样的探针时,将出现与分裂事件产生的两条染色体相对应的两条带。另一方面,如果没有发生分裂事件,则仅出现对应于完整染色体的一个条带。典型的结果如图3所示。
    6. 根据您使用的DNA标签试剂盒手册检测标记的探针。


      图3. CRISPR-PCS染色体分裂的典型结果。 IV由CRISPR-PCS在位置999,122.5处分裂。探针被设计为延伸到分裂点(从998,717到999,302)。野生型菌株(称为WT)显示对应于完整Chr的单条带。 IV,而转化体(指定为1,2和3)显示两条带,表明Chr。 IV在目标点分裂。

数据分析

每个实验中转化体的数量不同。通常,当仅靶向一个基因组位点时,可以获得数百个或更多的转化体,并且几乎所有转化体都具有预期的分裂。即使定位多个网站,也可以获得数百个转化体,尽管预期分裂的转化体的频率降低。对转化体的检查应产生10个转化体中至少一个正确分裂的转化体。

食谱

  1. LB板
    2%LB肉汤(Sigma-Aldrich)
    1.5%琼脂
  2. 酵母最低培养基(SD培养基)
    2%葡萄糖
    0.67%不含氨基酸的酵母氮碱(例如,BD Difco)
    注意:如果需要,可以使用含有用于营养缺陷选择的缺乏合适氨基酸的所有氨基酸的混合物。
    用NaOH调节至pH = 6.0 对于平板培养基,添加2%琼脂
  3. YPD媒体
    2%葡萄糖
    2%蛋白胨(例如,BD Bacto TM )
    1%酵母提取物(例如,BD Bacto TM)/
    用HCl调节至pH = 6.0

致谢

这项工作得到科学研究资助(B)[15H04475](SH),"挑战性探索性研究补助金"[26660066](至SH)和青年科学家助学金的支持(B)[15K18672](来自YS)来自日本的教育,文化,体育和科技部(MEXT)。

参考

  1. Agmon,N.,Pur,S.,Liefshitz,B.and Kupiec,M。(2009)。  同源重组期间修复机制选择的分析。 Nucleic Acids Res 37(15):5081-5092。
  2. Gietz,RD和Schiestl,RH(2007)。高使用LiAc/SS载体DNA/PEG方法的酵母菌转化效率。 Nat Protoc 2(1):31-34。
  3. Guldener,U.,Heck,S.,Fielder,T.,Beinhauer,J.and Hegemann,JH(1996)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm .nih.gov/pubmed/8692690"target ="_ blank">一种用于萌芽酵母重复使用的新型有效的基因破坏盒。核酸研究24(13):2519-2524 。
  4. 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。
  5. Li,MZ and Elledge,SJ(2012)。  SLIC :用于顺序和连接非依赖性克隆的方法。 Methods Mol Biol 852:51-59。
  6. Southern,E.(2006)。 Southern印迹。 Nat Protoc 1:518-525。
  7. Sugiyama,M.,Ikushima,S.,Nakazawa,T.,Kaneko,Y.and Harashima,S。(2005)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/16018552"target ="_ blank"> PCR介导的酿酒酵母重复染色体分裂。 38(6): 909-914。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sasano, Y. and Harashima, S. (2017). CRISPR-PCS Protocol for Chromosome Splitting and Splitting Event Detection in Saccharomyces cerevisiae. Bio-protocol 7(10): e2306. DOI: 10.21769/BioProtoc.2306.
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