Using CRISPR/Cas9 for Large Fragment Deletions in Saccharomyces cerevisiae

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Analytical Biochemistry
Jul 2016



CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) systems have emerged as a powerful tool for genome editing in many organisms. The wide use of CRISPR/Cas9 systems may be due to the fact that these systems contain a simple guide RNA (sgRNA) that is relatively easy to design and they are very versatile with the ability to simultaneously target multiple genes within a cell (Varshney et al., 2015). We have developed a CRISPR/Cas9 system to delete large genomic fragments (exceeding 30 kb) in Saccharomyces cerevisiae. One application of this technology is to study the effects of large-scale deletions of non-essential genes which may give insight into the function of gene clusters within chromosomes at the molecular level. In this protocol, we describe the general procedures for large fragment deletion in S. cerevisiae using CRISPR/Cas9 including: how to design CRISPR arrays and how to construct Cas9-crRNA expression plasmids as well as how to detect mutations introduced by the system within S. cerevisiae cells.

Keywords: CRISPR/Cas9 system (CRISPR/Cas9系统), Large fragment deletion (大片段缺失), Saccharomyces cerevisiae (酿酒酵母)


The CRISPR/Cas9 system is a rapid, efficient, low-cost, and versatile method for genome editing that can be applied in the fields of biology, agriculture, and medicine. To date, several protocols have been reported on how to make large-scale deletions within genomes. Each of these methods contains its own unique characteristics and advantages. The recently developed CRISPR/Cas9 system for excising large stretches of chromosomes has potential advantages over other methods such as the Latour system (Hirashima et al., 2006). The CRISPR/Cas9 system requires two components: (1) the Cas9 endonuclease for DNA cleavage and (2) a variable guide RNA (gRNA) that directs the Cas9 enzyme in a DNA sequence-specific manner (Cong et al., 2013). When Cas9 is targeted to a genomic locus by a gRNA, Cas9 initiates a DSB. The cell will respond to the DSB by repairing the damage via one of two major pathways: high-fidelity homology-dependent repair or error-prone non-homologous end joining (NHEJ).

The CRISPR/Cas9 system described here requires four components: Cas9 endonuclease, CRISPR array, trans-activating crRNA (tracrRNA), and RNase III (this activity is present in the host cell). The CRISPR array is a genomic locus from which pre-crRNAs are transcribed. In this system, the CRISPR array, engineered on the pCRCT plasmid, expresses multiple spacers flanked by direct repeats driven by a single promoter. Cas9 cannot be targeted by crRNA alone, it requires a crRNA-tracrRNA duplex to target it to a specific site within the genome. Two DNA oligonucleotides that encode for spacer sequences interspaced by a direct repeat (DR) were directly synthesized. The formed dsDNA encoding the crRNA was cloned into a Cas9 expression vector. Once the desired plasmid is constructed, transform S. cerevisiae with it and screen the transformants to obtain mutants with large genomic fragment deletions. In this protocol, the deletion efficiency (10%) is lower than described for deletion of genomic fragments using CRISPR/Cas9 in rice (Zhou et al., 2014). There are two possible reasons. The first one is that the genome may be repaired more rapidly in S. cerevisiae in comparison with that in rice. Another one is a stronger selection pressure used for screening rice transformants. In rice, the selection marker for transformants is hygromycin B, while in yeast Uracil as a selection marker is employed. The stronger selection pressure possibly increase the plasmid copy numbers in rice cells.

Materials and Reagents

  1. Escherichia coli: DH5ɑ (sanyou Biopharmaceuticals)
  2. S. cerevisiae strain: W303 (MATa ura3-52)
  3. pCRCT plasmid (Addgene, catalog number: 60621 )
  4. Salmon DNA (Sigma-Aldrich, catalog number: D1626 )
  5. Restriction enzyme: 10 U/μl Bsal (Takara Bio)
  6. 10x buffer G (Takara Bio)
  7. Taq DNA polymerase (Takara Bio)
  8. Antibiotic: 100 µg/ml ampicillin (Siyao)
  9. Pure plasmid mini kit (CWBIO)
  10. Yeast Gen DNA Kit (CWBIO)
  11. DNA oligonucleotide primers (GENEWIZ)
  12. PEG: Poly (ethylene glycol), BioXtra avg. molecular weight 3,350 (Sigma-Aldrich, catalog number: P3640 )
    Note: This product has been discontinued.
  13. Yeast extract (Oxoid, catalog number: LP0021 )
  14. Tryptone (Oxoid, catalog number: LP0042 )
  15. Dextrose
  16. Agar (Solarbio, catalog number: A8190 )
  17. Sodium chloride (NaCl) (Tianjin Kemiou Chemical Reagent)
  18. Peptone
  19. Primers
  20. Glucose (Tianjin Kemiou Chemical Reagent)
  21. Adenine (Sigma-Aldrich)
  22. Lithium acetate dihydrate (Sigma-Aldrich, catalog number: L6883-1KG )
  23. Agarose gel recovery kit (Biomiga)
  24. YPD liquid media (see Recipes)
  25. LB plates (with appropriate antibiotics included) (see Recipes)
  26. YPDA liquid medium (see Recipes)
  27. SC medium without uracil (see Recipes)


  1. PCR machine (or similar) (Biometra, model: TPfofessional )
  2. 42 °C water bath (or similar) (XINBAO, catalog number: HH-501BS )
  3. DNA electrophoresis apparatus (or similar) (SIM International, model: BIO-PRO 200E , catalog number: 0401RHSI049)
  4. Microcentrifuge (SCILOGEX, model: D2012 )
  5. Incubator (or similar, capable of incubation of agar plates at 37 °C or 28 °C) (CIMO, model: DNP-III )


  1. Find non-essential S. cerevisiae genes (after deletion or inactivation of which will not result in lethality) with one of the following databases:

  2. Design two 20 nucleotide (nt) spacer sequences for the CRISPR array
    Design the two 20 nt spacer sequences for the CRISPR array with the online tool E-CRISP. The following factors should be considered when designing the spacer sequence:
    1. Spacer sequence length is generally 17-20 nt. The protospacer adjacent motif (PAM) for the Streptococcus pyogenes Cas9 (SpCas9) is 5’-NGG-3’.
    2. The spacer sequence may be located on the non-transcribed strand or the transcribed strand.
    3. The selected spacer sequence should be specific to avoid off-target effects.
      The tool E-CRISP helps design site-specific spacer sequences.

  3. Construct the crRNA-Cas9 plasmid
    1. The dsDNA insertion fragment for the crRNA transcript which contains two spacer sequences and two partial direct repeat sequences has to be synthesized (Figure 1).

      Figure 1. Schematic representation of the dsDNA used for crRNAs expression

    2. The dsDNA insert for crRNA transcription is digested using BsaI and cloned into the BsaI-digested pCRCT plasmid (Bao et al., 2015).
      1. DNA digestion reaction:
        30 ng of pCRCT plasmid DNA (or the dsDNA)
        10x buffer G:                                                                2 µl
        Bsal:                                                                             1 µl
        Add H2O to                                                                   20 µl
      2. Incubate at 37 °C for 3 h.
      3. Analyze the digestion reaction with 1% (or 2%) (w/v) agarose gel electrophoresis and recover the digested DNA using an agarose gel purification kit.
    3. Ligate the dsDNA insert for crRNA transcription into pCRCT plasmids
      1. The ligation reaction:
        BsaI digested plasmid:                                                     2 µl (20 ng)
        BsaI digested dsDNA insert for crRNA transcription:       0.3 µl (20-50 ng)
        Ligation mixture:                                                               2.5 µl
        Add H2O to                                                                       5 µl
      2. Incubate at 16 °C overnight.
    4. Transform E. coli DH5α competent cells with the ligation product.
      1. Add 5 μl ligation mix into ice-cold competent E. coli DH5α (100 μl).
      2. Incubate mixture on ice for 30 min and heat-shock it at 42 °C for 90 sec.
      3. Cool down on ice for 2 min.
      4. Add 700 μl LB medium (without antibiotics) and incubate at 37 °C and shake at 250 rpm for 45 min.
      5. Plate 200 μl bacteria suspension on LB plate containing 100 μg/ml ampicillin.
      6. Incubate overnight at 37 °C in an incubator.
      7. Run a colony PCR for colonies per transformed pCRCT with CasYZF/CasYZR primers.
        PCR protocol
        95 °C     5 min       1 cycle
        95 °C     30 sec
        55 °C     30 sec     30 cycles
        72 °C     1 min
        72 °C     5 min       1 cycle
        4 °C       Hold         1 cycle
      8. Verify the product by sequencing.
      9. Pick positive clone(s) in 5 ml LB medium with 100 µg/ml ampicillin and incubate for 16 h, at 37 °C, 200 rpm in a shaker.
    5. Extract plasmids from the transformed DH5α cells using the Pure Plasmid Mini Kit.
    6. Transform crRNA-Cas9 plasmids into S. cerevisiae competent cells using LiAc/SS carrier DNA/PEG method (Gietz et al., 2007).

  4. Verification of Cas9-crRNA mediated large fragment deletions in S. cerevisiae.
    1. To verify Cas9-crRNA mediated large fragment deletions, a pair of gene specific primers is required to amplify the targeted region.
    2. Pick positive clone(s) from selection plate in 5 ml SC medium without uracil and incubate at 28 °C and shake at 200 rpm for 4 d.
    3. Extract S. cerevisiae genomic DNA from step D2 using Yeast Gen DNA Kit.
    4. Perform PCR amplification using genomic DNAs extracted from transgenic S. cerevisiae with the following PCR procedure:
      Note: P1 and P2 primers (Figure 2, Table 1) are designed based on the sequence flanking the target region to amplify the junction sequence, and amplicon is possible across the ligated junction only when large deletion occurred. P3-P8 primers can confirm the result. The primers can be designed by Primer3 (length: 18-25, GC content: 40%-60%).
      PCR protocol
      95 °C     5 min     1 cycle
      95 °C     30 sec
      X °C       30 sec   30 cycles
      72 °C     N min
      72 °C     5 min     1 cycle
      4 °C       Hold      1 cycle
      Note: Annealing temperature (X) and extension time (N) depend on primers.

      Figure 2. Large fragment deletion by CRISPR/Cas9 as described in this protocol.

      See Table 1 for the sequences of P1-P8, gRNA1 and gRNA2

      Table 1. The sequences of P1-P8, gRNA1 and gRNA2

    5. The PCR product is verified by electrophoresis (Figure 3).

      Figure 3. Confirmation of the large chromosomal modifications. A. M, marker; lane 1, mutant; lane 2, control. B, C and D. The PCR product with primers P3-P4, P5-P6, P7-P8 in the target region, respectively, 1a, 1b, 1c, the parental strain. 2a, 2b, 2c, the deletant. 


In this protocol, the large-scale deletion efficiency was only 10%. The likely reason for this is that the DSB can be repaired via NHEJ, which typically results in small insertions and/or deletions (indels) at the site of the break. While the HDR pathway allows high fidelity and precise editing, the efficiency of large-scale deletion in S. cerevisiae could be improved if the target vector contains the homology arms of target gene.


Note: The solvent for the media is ddH2O.

  1. YPD liquid media
    1% (w/v) yeast extract
    2% (w/v) tryptone
    2% (w/v) dextrose
    Autoclave and store at 4 °C
  2. LB plates (with appropriate antibiotics included)
    1.5% (w/v) agar
    1% (w/v) tryptone
    0.5% (w/v) yeast extract
    1% (w/v) NaCl
  3. YPDA liquid medium
    2% (w/v) peptone
    2% (w/v) glucose
    1% (w/v) yeast extract
    0.005% (w/v) adenine
    Autoclave and store at 4 °C
  4. SC medium without uracil
    2% (w/v) glucose
    0.67% (w/v) yeast nitrogen base
    Autoclave and store at 4 °C


This work was supported by the open fund of Key laboratory (No. 3333112) and Bioengineering key discipline of Hebei Province.


  1. Bao, Z., Xiao, H., Liang, J., Zhang, L., Xiong, X., Sun, N., Si, T. and Zhao, H. (2015). Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae. ACS Synth Biol 4(5): 585-594.
  2. 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: 819-823.
  3. Gietz, R. D. and Schiestl, R. H. (2007). Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1): 1-4.
  4. Hirashima, K., Iwaki, T., Takegawa, K., Gigahama, Y. and Tohda, H. (2006). A simple and effective chromosome modification method for large-scale deletion of genome sequences and identification of essential genes in fission yeast. Nucleic Acids Res 34(2): e11.
  5. Varshney, G. K., Pei, W., LaFave, M. C., Idol, J., Xu, L., Gallardo, V., Carrington, B., Bishop, K., Jones, M., Li, M., Harper, U., Huang, S. C., Prakash, A., Chen, W., Sood, R., Ledin, J. and Burgess, S. M. (2015). High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9. Genome Res 25(7): 1030-1042.
  6. Zhou, H., Liu, B., Weeks, D. P., Spalding, M. H. and Yang, B. (2014). Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Res 42(17): 10903-10914.


CRISPR / Cas9(集群定期间隔的短回文重复/ CRISPR相关蛋白9)系统已经成为许多生物体中基因组编辑的有力工具。 CRISPR / Cas9系统的广泛使用可能是由于这些系统包含相对容易设计的简单导向RNA(sgRNA),并且它们具有同时靶向细胞内多个基因的能力非常通用的事实(Varshney 等,,2015)。我们开发了一种CRISPR / Cas9系统来删除酿酒酵母中的大型基因组片段(超过30 kb)。该技术的一个应用是研究非必需基因的大规模缺失的影响,这可以了解染色体内基因簇在分子水平上的功能。在这个协议中,我们描述了在 S中大片段删除的一般程序。包括:如何设计CRISPR数组,以及如何构建Cas9-crRNA表达质粒,以及如何检测系统内引入的突变。细胞。
【背景】CRISPR / Cas9系统是一种快速,高效,低成本,多用途的基因组编辑方法,可应用于生物学,农业学和医学领域。迄今为止,已经有几个方案报告了如何在基因组内进行大规模的缺失。这些方法中的每一种都包含其独特的特征和优点。最近开发的用于切除大片染色体的CRISPR / Cas9系统具有优于其他方法(例如Latour系统)(Hirashima等人,2006)的优势。 CRISPR / Cas9系统需要两个组分:(1)用于DNA切割的Cas9内切核酸酶和(2)以DNA序列特异性方式引导Cas9酶的可变引导RNA(gRNA)(Cong等, em>。,2013)。当Cas9通过gRNA靶向基因座时,Cas9引发DSB。通过两个主要途径之一修复损伤,细胞将响应DSB:高保真同源性依赖性修复或易出错的非同源末端连接(NHEJ)。
 这里描述的CRISPR / Cas9系统需要四个组分:Cas9内切核酸酶,CRISPR阵列,反式激活crRNA(tracrRNA)和RNA酶III(该活性存在于宿主细胞中)。 CRISPR阵列是从其中转录前crRNAs的基因组座位。在该系统中,在pCRCT质粒上工程化的CRISPR阵列表达由单个启动子驱动的直接重复序列的多个间隔区。 Cas9不能被crRNA单独靶向,它需要一个crRNA-tracrRNA双链体将其靶向到基因组内的特定位点。直接合成编码由直接重复(DR)间隔的间隔序列的两个DNA寡核苷酸。将编码crRNA的形成的dsDNA克隆到Cas9表达载体中。一旦构建所需的质粒,转化。并用筛选转化体获得具有大基因组片段缺失的突变体。在该方案中,缺失效率(10%)低于使用水稻中CRISPR / Cas9的基因组片段缺失所述(Zhou等人,2014)。有两个可能的原因。第一个是基因组可能在S中更快地修复。酿酒酵母与水稻相比。另一种是用于筛选水稻转化体的更强的选择压力。在水稻中,转化体的选择标记是潮霉素B,而在酵母中使用尿嘧啶作为选择标记。较强的选择压力可能增加水稻细胞中的质粒拷贝数。

关键字:CRISPR/Cas9系统, 大片段缺失, 酿酒酵母


  1. 大肠杆菌:DH5ɑ(sanyou Biopharmaceuticals)
  2. S上。酿酒酵母菌株:W303(MATa ura3-52)
  3. pCRCT质粒(Addgene,目录号:60621)
  4. 鲑鱼DNA(Sigma-Aldrich,目录号:D1626)
  5. 限制酶:10U /μlBsa l(Takara Bio)
  6. 10倍缓冲液G(Takara Bio)
  7. Taq DNA聚合酶(Takara Bio)
  8. 抗生素:100μg/ ml氨苄青霉素(Siyao)
  9. 纯质粒小套件(CWBIO)
  10. 酵母基因DNA试剂盒(CWBIO)
  11. DNA寡核苷酸引物(GENEWIZ)
  12. PEG:聚(乙二醇),BioXtra平均分子量3,350(Sigma-Aldrich,目录号:P3640)
  13. 酵母提取物(Oxoid,目录号:LP0021)
  14. 胰蛋白胨(Oxoid,目录号:LP0042)
  15. 葡萄糖
  16. 琼脂(Solarbio,目录号:A8190)
  17. 氯化钠(NaCl)(天津凯美化学试剂)
  18. 蛋白胨
  19. 引物
  20. 葡萄糖(天津凯美化学试剂)
  21. 腺嘌呤(Sigma-Aldrich)
  22. 乙酸锂二水合物(Sigma-Aldrich,目录号:L6883-1KG)
  23. 琼脂糖凝胶回收试剂盒(Biomiga)
  24. YPD液体介质(见配方)
  25. LB平板(含适当的抗生素)(参见食谱)
  26. YPDA液体介质(见配方)
  27. 没有尿嘧啶的SC培养基(参见食谱)


  1. PCR机器(或类似产品)(Biometra,型号:TPfofessional)
  2. 42℃水浴(或类似物)(XINBAO,目录号:HH-501BS)
  3. DNA电泳装置(或类似物)(SIM International,型号:BIO-PRO 200E,目录号:0401RHSI049)
  4. 微量离心机(SCILOGEX,型号:D2012)
  5. 孵育器(或类似的,能够在37℃或28℃孵育琼脂平板)(CIMO,型号:DNP-III)


  1. 查找非必需的 S。使用以下数据库之一进行基因(其删除或失活不会导致致死率): /

  2. 为CRISPR阵列设计两个20个核苷酸(nt)间隔序列 使用在线工具E-CRISP设计CRISPR阵列的两个20 nt间隔序列。设计间隔序列时应考虑以下因素:
    1. 间隔序列长度一般为17-20nt。用于化脓性链球菌Cas9(SpCas9)的原始相邻基序(PAM)是5'-NGG-3'。
    2. 间隔序列可以位于非转录链或转录链上
    3. 选择的间隔序列应该是特异性的,以避免脱靶效应。
      工具E-CRISP有助于设计特定于位置的间隔序列。 CRISP /

  3. 构建crRNA-Cas9质粒
    1. 必须合成含有两个间隔序列和两个部分直接重复序列的crRNA转录本的dsDNA插入片段(图1)。


    2. 使用Bsa I消化用于crRNA转录的dsDNA插入片段并克隆到BaxI消化的pCRCT质粒(Bao等人,2015)中,。
      1. DNA消化反应:
        30ng pCRCT质粒DNA(或dsDNA)
        10倍缓冲G:                                                                2 µl
        Bsal:                                                                             1 µl
        Add H2O to                                                                   20 µl
      2. 在37°C孵育3 h
      3. 用1%(或2%)(w / v)琼脂糖凝胶电泳分析消化反应,并使用琼脂糖凝胶纯化试剂盒回收消化的DNA。
    3. 将dsDNA插入片段连接到pCRCT质粒中
      1. 结扎反应:
        Bsa 我消化的质粒:                                                    2 µl (20 ng)
        BsaI digested dsDNA insert for crRNA transcription:       0.3 µl (20-50 ng)
        Ligation mixture:                                                               2.5 µl
        Add H2O to                                                                       5 µl
      2. 在16℃下孵育过夜。
    4. 变换E。 大肠杆菌DH5α感受态细胞与连接产物。
      1. 将5μl连接混合物加入冰冷的感受态细胞中。 大肠杆菌DH5α(100μl)。
      2. 将混合物在冰上孵育30分钟,并在42℃下热冲击90秒。
      3. 在冰上冷却2分钟。
      4. 加入700μlLB培养基(无抗生素),37℃孵育,250rpm摇动45分钟。
      5. 在含有100μg/ ml氨苄青霉素的LB平板上平板200μl细菌悬浮液。
      6. 在孵化器中37℃孵育过夜。
      7. 用CasYZF / CasYZR引物对转化的pCRCT进行菌落PCR进行菌落PCR PCR方案
        95 °C     30 sec
        55 °C     30 sec     30 cycles
        72 °C     1 min
        72 °C     5 min       1 cycle
        4 °C       Hold         1 cycle
      8. 通过排序验证产品。
      9. 在含有100μg/ ml氨苄青霉素的5ml LB培养基中挑取阳性克隆,并在37℃,200rpm在振荡器中孵育16小时。
    5. 使用纯质粒Mini Kit从转化的DH5α细胞中提取质粒
    6. 将crRNA-Cas9质粒转化成S。使用LiAc / SS载体DNA / PEG方法的酿酒酵母感受态细胞(Gietz等人,2007)。

  4. Cas9-crRNA介导的大片段缺失的验证。酿酒酵母。
    1. 为了验证Cas9-crRNA介导的大片段缺失,需要一对基因特异性引物来扩增靶向区域。
    2. 在不含尿嘧啶的5ml SC培养基中,从选择板中挑取阳性克隆,并在28℃下孵育并在200rpm下摇动4天。
    3. 提取 S。使用酵母基因DNA试剂盒的来自步骤D2的酿酒酵母基因组DNA。
    4. 使用从转基因S提取的基因组DNA进行PCR扩增。具有以下PCR程序的啤酒酵母:
      注意:基于靶区域侧翼的扩增连接序列的序列设计P 1和/或2> 2>引物(图2,表1)和扩增子只有在发生大量缺失时,才可能穿过结扎结。 P 3 8'引物可以证实结果。引物可以由Primer3设计(长度:18-25,GC含量:40%-60%)。
      95 °C     5 min     1 cycle
      95 °C     30 sec
      X °C       30 sec   30 cycles
      72 °C     N min
      72 °C     5 min     1 cycle
      4 °C       Hold      1 cycle

      图2.本协议中描述的CRISPR / Cas9的大片段删除。

      参见表1,以获得P 1-Sub 8,gRNA1和gRNA2的序列

      表1. P1-P8,gRNA1和gRNA2的序列

    5. 通过电泳验证PCR产物(图3)

      图3.确认大染色体修饰 A. M,标记;泳道1,突变体;车道2,控制。 B,C和D。具有引物P 3 - 3 - 4,P 5 - 6 - 6的PCR产物,目标区域中的第7〜第_ 8个子区域,1a,1b,1c,亲本菌株。 2a,2b,2c,删除。 




注意:介质溶剂为ddH 2 O。

  1. YPD液体介质
    1%(w / v)酵母提取物 2%(w / v)胰蛋白胨
    2%(w / v)葡萄糖
  2. LB板(含适当的抗生素)
    1.5%(w / v)琼脂
    1%(w / v)胰蛋白胨
    0.5%(w / v)酵母提取物 1%(w / v)NaCl
  3. YPDA液体介质
    2%(w / v)蛋白胨
    2%(w / v)葡萄糖
    1%(w / v)酵母提取物 0.005%(w / v)腺嘌呤
  4. 没有尿嘧啶的SC培养基
    2%(w / v)葡萄糖
    0.67%(w / v)酵母氮碱 高压灭菌并储存在4°C




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引用:Hao, H., Huang, J., Liu, T., Tang, H. and zhang, L. (2017). Using CRISPR/Cas9 for Large Fragment Deletions in Saccharomyces cerevisiae. Bio-protocol 7(14): e2415. DOI: 10.21769/BioProtoc.2415.