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Jan 2017

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Single and Multiplexed Gene Editing in Ustilago maydis Using CRISPR-Cas9
使用CRISPR-Cas9对玉米黑粉菌进行单个和多个基因的编辑   

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Abstract

The smut fungus Ustilago maydis is an established model organism for elucidating how biotrophic pathogens colonize plants and how gene families contribute to virulence. Here we describe a step by step protocol for the generation of CRISPR plasmids for single and multiplexed gene editing in U. maydis. Furthermore, we describe the necessary steps required for generating edited clonal populations, losing the Cas9 containing plasmid, and for selecting the desired clones.

Keywords: CRISPR-Cas9 (CRISPR-Cas9), Multiplexing (多重), Gene editing (基因编辑), Ustilago maydis (玉米黑粉菌), Smut fungi (黑粉菌)

Background

The basidiomycete fungus U. maydis is a model organism that has enabled important discoveries in topics like DNA repair and homologous recombination, mating, sexual development, secondary metabolism, filamentous growth, RNA transport and virulence (Holliday, 2004; Steinberg, 2007; Bakkeren et al., 2008; Holloman et al., 2008; Lanver et al., 2017; Niessing et al., 2018). U. maydis is a biotrophic plant pathogen that causes smut disease on maize. As a typical member of the large group of smut fungi its sexual development is intimately coupled with its ability to colonize plants. The popularity of U. maydis as a model organism resides in its short pathogenic life cycle that is completed in less than two weeks, its accessibility for forward and reverse genetics that includes the establishment of self-replicating plasmids (Tsukuda et al., 1988), the development of solopathogenic strains able to cause disease without mating and an available well-annotated genome (Kämper et al., 2006). In U. maydis, homologous recombination-based genome manipulations are very efficient, but become tedious when several genes have to be modified because the number of selectable markers is limited (Schuster et al., 2016). The most recent addition to the technical toolboxes for this organism has been the adaptation of the CRISPR-Cas9 genome editing technology (Schuster et al., 2016) and its development for multiplexed editing (Schuster et al., 2018). The CRISPR-Cas9 genome editing technology, developed from research in bacterial immune systems (Barrangou et al., 2007), has revolutionized molecular biology. The system allows efficient modification of almost any given sequence (Jinek et al., 2012) and has been established for a large number of organisms including filamentous fungi (Deng et al., 2017; Shi et al., 2017).

The CRISPR-Cas9 technology established for U. maydis is based on an all-in-one plasmid approach. A self-replicating plasmid (Tsukuda et al., 1988) is used as a backbone to provide the U. maydis codon-optimized Cas9 gene and the U6 promoter for sgRNA expression. This plasmid is unstable without selection and is rapidly lost (Figure 1 and Schuster et al. [2016]). The system proved very efficient for disruption of single genes reaching on average 70% of edited cells in progeny of one transformant (Schuster et al., 2016). A multiplexing version of the system was generated by using tRNA promoter-based expression cassettes that enabled the expression of several sgRNAs from the same plasmid (Schuster et al., 2018). This modification combined with elevated and longer expression of the Cas9 protein was used for the simultaneous disruption of five effector genes with 70% efficiency (Schuster et al., 2018). The multiplexed version of CRISPR-Cas9 has also been successfully applied for elucidating the contribution of oligopeptide transporters to virulence (Lanver et al., 2018).

Materials and Reagents

  1. Consumables
    1. Sterile pipette tip
    2. Toothpick
    3. 60 mm x 15 mm round Petri dishes
  2. Competent cells
    E. coli competent cells e.g., One Shot TOP10 Chemically Competent E. coli (Thermo Fisher Scientific, catalog number: C404010 )
  3. Plasmids and double-stranded DNA
    1. pCas9_sgRNA_0 (Addgene, catalog number: 70763 or available on request) (Figure 1A)
    2. pMS73 (Addgene, catalog number: 110629 or available on request) (Figure 1B)
    3. Double-stranded DNA fragments can be purchased e.g., gBlocks (Integrated DNA Technologies) or GeneStrands (Eurofins Genomics)
  4. Oligonucleotides 10 pmol/µl
    1. oMS59: 5’ ATTCGTGATTTACACCAAACACGC 3’
    2. oMS49: 5’ CCCCTCGTCTCGCGCCTCATTGGTCGAATTG 3’
    3. oSR224: 5’ CTACACTCAGCACACGATGT 3’
  5. Enzymes and buffers
    1. Acc65I restriction endonuclease (New England Biolabs, catalog number: R0599S )
    2. Gibson Assembly® master mix (New England Biolabs, catalog number: E2611S )
    3. Phusion High-Fidelity DNA Polymerase (New England Biolabs, catalog number: M0530S ) or BioMix Red (Bioline, catalog number: BIO-25006 )
  6. Kits
    1. QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27104 )
    2. Wizard SV Gel and PCR Clean-Up system (Promega, catalog number: A9281 )
    3. Optional: Plate Seq Kit PCR (Eurofins genomics)
  7. Antibiotics
    1. Ampicillin 10 mg/ml stock in Milli-Q water (Carl Roth, catalog number: HP62.1 )
    2. Carboxin 5 mg/ml stock in methanol (Sigma-Aldrich, catalog number: 45371 )
  8. Reagents
    1. Tryptone (BD Biosciences)
    2. Yeast extract (BD Biosciences)
    3. Sodium chloride (NaCl)
    4. Peptone (BD Biosciences)
    5. Sucrose (BD Biosciences)
    6. Sorbitol (Sigma-Aldrich, catalog number: S1876 )
    7. Bacto agar (BD Biosciences)
    8. Potato Dextrose Broth (BD Biosciences)
  9. Media (see Recipes)
    1. Yeast extract tryptone (dYT) liquid medium + 100 µg/ml ampicillin
    2. YT agar plates + 100 µg/ml ampicillin
    3. YEPS light liquid medium +/- 2 µg/ml carboxin
    4. Two layered regeneration agar light (RegAgar) plates (Bottom layer (10 ml) with 4 µg/ml carboxin and top layer (10 ml) without carboxin (Prepare fresh))
    5. Potato dextrose (PD) agar plates +/- 2 µg/ml carboxin
    Note: All media need to be autoclaved. For plates, 20 ml of media should be used per Petri dish.

Equipment

Note: No equipment from specific manufacturers is required. Any equivalent device can be used.

  1. Pipettes
  2. Computer with internet access
  3. Plate incubators (28 °C, 37 °C)
  4. Shakers (28 °C, 37 °C)
  5. ThermoMixer (e.g., Eppendorf, model: ThermoMixer® C , catalog number: 5382000015)
  6. Thermocycler (e.g., Analytik, Jena, Biometra, catalog number: 846-2-070-301 )
  7. Tabletop centrifuge (e.g., Thermo Fisher Scientific, catalog number: 75008801 )

Software

No specific software is required. Any appropriate alignment program like CloneManager 9 Professional Edition (Scientific & Educational Software) or CLC Main Workbench (QIAGEN) can be used.

Procedure

  1. Select target sequences with E-CRISP (Heigwer et al., 2014)
    Note: Although several tools have been developed for sgRNA design, our experience is limited to the E-CRISP tool that proved to be very reliable.
    1. Select U. maydis as the organism in the E-CRISP webpage (http://www.e-crisp.org/E-CRISP/).
    2. Click on the ‘Enter target sequence’ box and insert target genomic DNA sequence in FASTA format.
      Note: Use the coding sequence of the gene you want to inactivate and restrict your search to the most 5´ end of the gene. In case you do not find a suitable sgRNA you can expand the input sequence towards the 3´end.
    3. Click on ‘display advanced options’ on the ‘Start application’ panel and make sure the 3’ PAM is NGG.
      Notes:
      1. The E-CRISP tool offers to restrict the search of target sequences to those starting with a G. However, this restriction is not necessary when using our plasmid backbones, because our linearized plasmids retain a G overhang at the 3´ end of the U6 promoter that serves as the first G preferred for initiating transcription at the U6 promoter.
      2. The E-CRISP tool gives you the option to design truncated target sequences (Fu et al., 2014). We usually use target sequences of 20 nt but have been successful using target sequences as short as 18 nt.
    4. Click on ‘Start sgRNA search’.
    5. Select the target sequence with the highest score and most 5’ in the gene. Double check that the target gene(s) correspond to the gene(s) you want to disrupt.
  2. Design sgRNA expression cassettes following examples in Table 1 and insert them into pCas9_sgRNA_0 or pMS73 (Figure 1)
    Note: The CRISPR-Cas9 plasmids developed for genome editing in U. maydis confer ampicillin resistance in E. coli and carboxin resistance in U. maydis. These plasmids contain a U. maydis ARS element for autonomous replication (Tsukuda et al., 1988). The plasmid backbones also contain the U. maydis U6 promoter just before an Acc65I site that is used for integration of the sgRNA expression cassette via Gibson Assembly® (Figure 1). Expression of the codon-optimized Cas9 is driven by the constitutive otef promoter in pCas9_sgRNA_0 (Figure 1A) or by the stronger hsp70 promoter in pMS73 (Figure 1B).


    Figure 1. Scheme of the CRISPR-Cas9 plasmids and cloning strategy. A. Schematic representation of pCas9_sgRNA_0 and cloning strategy for the introduction of a single sgRNA. A user-defined targeting fragment can be cloned into the Acc65I-linearized pCas9_sgRNA_0 backbone via Gibson Assembly®. B. Schematic representation of pMS73 and cloning strategy for the introduction of two or more sgRNA expression cassettes. User-defined left and right border cassettes and a variable number of middle cassettes can be cloned into the Acc65I-linearized pMS73 backbone via Gibson Assembly®. The target sequences are used as Gibson overhangs and the number of middle cassettes can be modified depending on the number of target sequences that need to be inserted. ori: Origin of replication, bla: β-lactamase gene that confers resistance to ampicillin in E. coli, ARS: autonomously replicating sequence for replication in U. maydis, ipR: U. maydis ip allele that confers resistance to carboxin. Numbers in yellow boxes indicate different targeting sequences. The scaffold sequence corresponds to the minimal scaffold required for Cas9 binding (Hsu et al., 2013). tRNA promoters used to drive expression of different sgRNAs are indicated by differently colored arrowheads. Cyan line indicates the Gibson overhang to PU6. Green line indicates the Gibson overhang to Potef. Purple line indicates the Gibson overhang to Phsp70.

    1. Introduce your target sequences in the appropriate template (Table 1).
    2. Purchase the designed sequences as double-stranded DNA fragments.
      Note: An alternative to purchasing the sgRNA expression cassettes is their generation by PCR amplification from a template. You can use double-stranded DNA fragments as templates for such a PCR.

      Table 1. Templates for generating double-stranded DNA fragments containing the target sequences of interest

      *Colors in Table 1 correspond to those in Figure 1.
      **The scaffold sequence corresponds to the minimal scaffold required for Cas9 binding (Hsu et al., 2013).

    Note: The sequences labeled as tRNA promoter contain both the DNA sequence of the tRNA ending up in the mature tRNA and 100 bp upstream from the part encoding the mature tRNA.

  3. Cloning of the sgRNA expression cassettes into the CRISPR plasmids via Gibson Assembly® (Gibson et al., 2010)
    1. Linearize pCas9_sgRNA_0 or pMS73 with Acc65I.
    2. Clone the sgRNA expression cassettes into the backbone via Gibson Assembly® according to the manufacturer’s protocol.
      Note, we have used Gibson Assembly® Master Mix (Instruction Manual, NEB #E2611S/L 10/50 reactions, Version 4.0).
    3. Transform part of the Gibson Assembly® reaction into competent E. coli cells according to the manufacturer’s protocol and select for growth on YT plates containing 100 µg/ml ampicillin. Incubate the selection plates overnight at 37 °C.
      Note: We have used the heat shock E. coli transformation protocol (Bacterial Transformation Workflow–4 Main Steps) as given at Thermo Fischer website.
    4. Pick some colonies to check for the correct insertion of sgRNA expression cassettes. Use a sterile pipette tip or toothpick to inoculate a single colony into 3 ml of dYT medium with 100 µg/ml ampicillin. Incubate with shaking at 37 °C overnight.
    5. Isolate the plasmid DNA from bacterial cultures by using e.g., a QIAprep spin miniprep kit according to the manufacturer’s instructions.
    6. Test for plasmids with inserts by restriction analysis and sequencing.
    Notes:
    1. For assessing the successful insertion of only one cassette, it is normally sufficient to test plasmids from two transformants or go directly for sequencing. If more than one cassete is to be inserted, it is usually sufficient to analyze plasmids from six transformants by restriction analysis first and follow candidates up by sequencing. When restriction analyses are performed, enzymes should be chosen which cleave outside the cassette(s) and allow to estimate the number of cassettes inserted.
    2. For plasmid sequencing use the U6-Fwd primer oMS59: ATTCGTGATTTACACCAAACACGC and the reverse primer Potef-Rv oMS49: CCCCTCGTCTCGCGCCTCATTGGTCGAATTG for pCas9_sgRNA_0 derived plasmids or oMS59: ATTCGTGATTTACACCAAACACGC and Phsp70-Rv oSR224: CTACACTCAGCACACGATGT for pMS73-derived plasmids. 
  4. Transformation of the CRISPR-Cas9 plasmid in U. maydis protoplasts
    1. Transform 50 µl of U. maydis protoplasts with no more than 500 ng of the pCas9_sgRNA_0 derived or pMS73 derived plasmids.
    2. Plate the transformation mixture on a freshly prepared two-layered RegAgar plate containing 4 µg/ml carboxin in the bottom layer and no carboxin in the top layer and incubate four days at 28 °C until transformants are clearly visible. 
    Notes:
    1. Transformation of the CRISPR-Cas9 constructs into U. maydis follows the protocol developed by Schulz et al. (1990).
    2. Note: See Bösch et al. (2016) for a detailed description of the protocol for the generation of protoplasts and the polyethylene glycol (PEG)-mediated transformation.
  5. Optional: Transfer single colonies from the transformation plate to 3 ml liquid YEPSL medium containing carboxin (2 µg/ml) to increase CRISPR-Cas9 efficiency.
    1. Incubate the culture and shake it at 200 rpm and 28 °C overnight.
    2. Dilute the cultures 1:100 into fresh selection medium and allow them to grow until saturation (approximately 20 h). Repeat three times.
    Note: CRISPR-Cas9-mediated genome editing in U. maydis is very efficient (Schuster et al., 2016). This step is only necessary when three sgRNAs or more are used simultaneously or a chosen sgRNA works inefficiently.
  6. Isolation of colonies with a defined editing event
    1. Streak out 10-12 transformants (in case of single gene disruption), or more (in case of multiple gene disruptions) from the original transformation plate (Step 4b) on PD plates without carboxin, or in the case of incubation in liquid medium (Step 5b), spot 1 µl of cells on a PD plate without carboxin and streak them out .
    2. Incubate the plate for two days at 28 °C.
    3. Generate a master plate to store colonies to be tested. To do this, pick single colonies with a flat toothpick and transfer to a new PD agar plate. Spread the transferred cells to obtain a larger patch in which cells grow up to form a colony and arrange these using a grid pattern for easier later identification. Incubate overnight at 28 °C (Figure 2).
    Notes:
    1. This step is of crucial importance because only in the absence of carboxin will the Cas9-containing plasmid be lost. Fungal colonies and cultures grown under selection pressure are heterogeneous populations: a colony (or culture grown with carboxin selection) will contain cells with independent editing events depending on the generation in which editing occurred - except when the CRISPR event took place in the initially transformed cell.
    2. At this step, you can confirm the loss of the CRISPR-Cas9 plasmid (see Step 8) by replica-plating the single colonies on a PD-carboxin plate in parallel to generate the master plate and selecting those colonies from the master plate which are unable to grow on the carboxin plate.


      Figure 2. Example of a master plate containing colonies to be tested. Generation of a master plate is advisable to facilitate the screening process. Single colonies derived from individual U. maydis transformants were transferred to a new PD plate following a grid pattern and labeled with numbers for identification. Transfers were made with flat toothpicks, cells were placed in the center of a grid square and slightly spread to increase the area in which cells can grow up and form a colony. A. Master plate before incubation; B. Master plate after incubation overnight at 28 °C.

  7. Colony PCR-based screen for CRISPR-Cas9 induced mutations
    1. Design primers enabling the amplification of a PCR product of about 500 bp containing the target site.
    2. With a sterile pipet tip or a toothpick, pick a small amount of a colony from the master plate (Step 6c) and place it in a well of a 96-well plate containing 20 µl of 20 mM NaOH.
    3. Shake the plate for 30 min at room temperature at 800 rpm using e.g., a ThermoMixer.
    4. Set up a 20 µl PCR reaction using a high fidelity polymerase e.g., Phusion DNA Polymerase or BioMix Red as suggested by the manufacturer. Use 1 µl of the lysed cells as DNA template.
      Note: In case of multiplexing, a separate PCR for each target site needs to be performed. If you have many colonies and/or sites to test, we recommend using kits like the Plate Seq Kit PCR. In this case, perform the PCR reaction in this step directly in the 96-well-PCR-plate provided in the kit.
    5. Amplify the PCR products in a Thermocycler following the polymerase manufacturer’s instructions.
    6. Sequence the PCR fragment either after cleanup with e.g., the Wizard SV Gel and PCR cleanup System or e.g., in the Plate Seq Kit from Eurofins.
  8. Loss of the CRISPR-Cas9 plasmid
    1. If you have not screened for the loss of the plasmid before (see note after Step 6), pick some cells with a toothpick from the master plate and streak out on a PD plate containing carboxin. Incubate for two days at 28 °C. Go back to the master plate and use colonies that were unable to grow on the carboxin plate, these have lost the plasmid.
      Note: We observe retention of the plasmid in colonies on the master plate in about 5% of the cases.
    2. Growth on the carboxin plate means that some cells still contain the plasmid. If this is the case and it is this colony that contains the desired mutation, inoculate cells from this strain from the master plate in 3 ml YESPSL medium without carboxin and grow overnight at 28 °C.
    3. Take cells from this overnight culture and spread on a PD plate without carboxin to obtain single colonies. Incubate for two days at 28 °C.
    4. Replica-plate single colonies on both, a PD plate with carboxin and a PD plate without carboxin, and incubate overnight at 28 °C. Select colonies that have lost the plasmid i.e., are unable to grow on the plate containing carboxin.
    5. If necessary, repeat the loss of plasmid procedure until there is no growth on selection plates.
    Note: Resequence the final clone to verify that you have obtained the desired mutation.

Data analysis

The sequencing data should be analyzed by alignment against the wild-type sequence using software like CloneManager or CLC Main Workbench. Frameshifts mutations are generated when the number of nucleotides deleted or inserted cannot be divided by three. Alternatively, indels that generate a premature stop codon can be used. It is mandatory to inspect the sequencing chromatograms carefully and check for the presence of overlapping peaks. Overlapping peaks are a sign of genetic heterogeneity in the sequenced sample (Figure 3).


Figure 3. Representative sequencing results. Alignment of nucleotide sequences and chromatograms of a wild-type (WT) allele, a corresponding edited allele lacking 19 bp and a heterogenic sample from the same experiment. Note that a sequence is provided (dark orange) by the sequencing company for the heterogenic sample taking into account the highest peaks and incorrectly suggesting sequence homogeneity. Heterogeneity can only be recognized by careful analysis of the chromatogram which in this case shows that the sequence up to the arrow (predicted Cas9 cleavage site) is homogeneous and becomes heterogeneous from this point onwards. Target sequence and PAM sequence are depicted as colored boxes in the wild-type sequence. The alignment was generated with CLC Main Workbench.

Notes

  1. The CRISPR-Cas9 system in U. maydis has proven to be highly efficient for the disruption of single genes where up to 100% of the tested transformants contained edited cells (Schuster et al., 2016). For sgRNA with lower editing efficiency, the efficiency could be improved by higher expression of the Cas9 protein using a stronger promoter (i.e., Phsp70 instead of Potef) and by extending exposure to Cas9 by maintaining the transformed cells in liquid culture under conditions selecting for the Cas9 expressing plasmid (Schuster et al., 2018). Using longer Cas9 exposure times, we have been able to improve editing using numerous sgRNAs. We have rarely been confronted with target sequences that either do not work at all or that give very low gene editing efficiency. In such cases, changing the target sequence provides an efficient solution.
  2. Two independently edited strains have been analyzed for off-target mutations and no off-targets where found via whole genome sequencing (Schuster et al., 2016). Since off-targets will be specific for each sgRNA, we recommend generating three independent mutants in a given gene or group of genes. If these mutants all show the same phenotypic alteration and this phenotype can be complemented by introducing the wild-type gene or genes, you have convincing evidence that the phenotype is caused by the generated mutation(s).
  3. The genomic location of genes to be edited should be taken into account when designing a multiplexing experiment. Two or more simultaneous CRISPR events in the same chromosome could lead to undesired chromosomal deletions or rearrangements. In such cases, sequential editing should be performed.

Recipes

  1. Yeast extract tryptone (dYT) medium
    This medium consists of:
    1.6% (w/v) tryptone (BD Biosciences)
    1% (w/v) yeast extract (BD Biosciences)
    0.5% (w/v) NaCl in double distilled water
    Note: Autoclave and store at room temperature before use.
  2. Yeast extract tryptone (YT) agar medium
    This medium consists of:
    0.8% (w/v) tryptone (BD Biosciences)
    0.5% (w/v) yeast extract (BD Biosciences)
    0.5% (w/v) NaCl
    1.3% Bacto agar (BD Biosciences) in double distilled water
    Note: Autoclave and immediately pour into Petri dishes (20 ml per Petri dish).
  3. YEPSL liquid medium
    This medium consists of:
    0.4% (w/v) yeast extract (BD Biosciences)
    0.4% (w/v) peptone (BD Biosciences)
    2% (w/v) sucrose (BD Biosciences) in double distilled water
    Note: Autoclave and store at room temperature before use.
  4. Regeneration agar light (RegAgar)
    This medium consists of:
    1% (w/v) yeast extract (BD Biosciences)
    0.4% (w/v) peptone (BD Biosciences)
    0.4% (w/v) sucrose (Roth)
    18.22% (w/v) sorbitol (Sigma)
    1.5% Bacto agar (BD Biosciences) in double distilled water
    Notes:
    1. Autoclave and store at room temperature before use.
    2. During U. maydis transformation, the agar is melted in a microwave oven, mixed well and cooled down to 50-60 °C. Carboxin (4 µg/ml final concentration) is added and 10 ml of this supplemented RegAgar is poured into a Petri dish. Once this is solidified, an additional 10 ml of molten RegAgar without carboxin is poured as a top layer into the same Petri dish. This second layer should be poured about 25-30 min before plating the transformation mixture (see transformation protocol by Bösch et al., 2016). 
  5. Potato dextrose (PD) agar medium
    This medium consists of:
    2.4% Potato Dextrose Broth (BD Biosciences)
    2% Bacto agar (BD Biosciences) in double distilled water
    Note: Autoclave and immediately pour into Petri dishes (20 ml per Petri dish).

Acknowledgments

This work was funded by the Max Planck Society. The protocol is adapted from Schuster et al. (2016 and 2018). We would like to acknowledge present and past members of the Kahmann Lab who use CRISPR-Cas9 mediated genome editing regularly and provided feedback on the protocol. The authors declare that there are no conflicts of interest nor competing interests related to this article.

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  19. Shi, T. Q., Liu, G. N., Ji, R. Y., Shi, K., Song, P., Ren, L. J., Huang, H. and Ji, X. J. (2017). CRISPR/Cas9-based genome editing of the filamentous fungi: the state of the art. Appl Microbiol Biotechnol 101(20): 7435-7443.
  20. Steinberg, G. (2007). Tracks for traffic: microtubules in the plant pathogen Ustilago maydis. New Phytol 174(4): 721-733.
  21. Tsukuda, T., Carleton, S., Fotheringham, S. and Holloman, W. K. (1988). Isolation and characterization of an autonomously replicating sequence from Ustilago maydis. Mol Cell Biol 8(9): 3703-3709.

简介

黑穗病真菌 Ustilago maydis 是一种既定的模式生物,用于阐明生物营养病原体如何在植物中定殖以及基因家族如何对毒力作出贡献。 在这里,我们描述了用于生成用于 U中单个和多重基因编辑的CRISPR质粒的逐步方案。玉米小斑病。 此外,我们描述了产生编辑的克隆群体,丢失含有Cas9的质粒以及选择所需克隆所需的必要步骤。

【背景】担子菌真菌 U. maydis 是一种模式生物,能够在DNA修复和同源重组,交配,性发育,次级代谢,丝状生长,RNA转运和毒力等主题中获得重要发现(Holliday,2004; Steinberg,2007; Bakkeren et al。,2008; Holloman et al。,2008; Lanver et al。,2017; Niessing et al。 ,2018年)。 Ú。 maydis 是一种生物营养的植物病原体,可引起玉米的黑穗病。作为大群黑穗病真菌的典型成员,其性发育与其定殖植物的能力密切相关。 U的受欢迎程度。 maydis 作为一种模式生物存在于其短暂的致病生命周期中,该生命周期在不到两周内完成,其可用于正向和反向遗传,包括建立自我复制的质粒(Tsukuda 等。,1988),能够在没有交配的情况下引起疾病的致病性病毒株的开发和可用的注释良好的基因组(Kämper et al。,2006)。在 U. maydis ,基于同源重组的基因组操作非常有效,但由于选择标记的数量有限,因此必须修改几个基因时变得乏味(Schuster et al。,2016)。该生物体技术工具箱的最新成员是CRISPR-Cas9基因组编辑技术的改编(Schuster et al。,2016)及其多重编辑的发展(Schuster et al。,2018)。从细菌免疫系统研究开发的CRISPR-Cas9基因组编辑技术(Barrangou et al。,2007),彻底改变了分子生物学。该系统允许对几乎任何给定序列进行有效修饰(Jinek et al。,2012),并已建立用于包括丝状真菌在内的大量生物体(Deng et al。 ,2017; Shi et al。,2017)。

为 U建立的CRISPR-Cas9技术。 maydis 基于一体化质粒方法。使用自我复制质粒(Tsukuda et al。,1988)作为骨架来提供 U. maydis 密码子优化的Cas9基因和用于sgRNA表达的U6启动子。该质粒在没有选择的情况下是不稳定的并且迅速丢失(图1和Schuster 等人 [2016])。该系统被证明非常有效地破坏单个基因,在一个转化体的后代中平均达到70%的编辑细胞(Schuster et al。,2016)。通过使用能够从相同质粒表达几种sgRNA的基于tRNA启动子的表达盒产生该系统的多重形式(Schuster 等,,2018)。这种修饰与Cas9蛋白的升高和更长的表达相结合,用于同时破坏效率为70%的效应基因(Schuster et al。,2018)。 CRISPR-Cas9的多重版本也已成功应用于阐明寡肽转运蛋白对毒力的贡献(Lanver et al。,2018)。

关键字:CRISPR-Cas9, 多重, 基因编辑, 玉米黑粉菌, 黑粉菌

材料和试剂

  1. 耗材
    1. 无菌移液器吸头
    2. 牙签
    3. 60毫米x 15毫米圆形培养皿
  2. 主管细胞
    电子。大肠杆菌感受态细胞例如,One Shot TOP10化学感受器 E.大肠杆菌(赛默飞世尔科技,目录号:C404010)
  3. 质粒和双链DNA
    1. pCas9_sgRNA_0(Addgene,目录号:70763或根据要求提供)(图1A)
    2. pMS73(Addgene,目录号:110629或根据要求提供)(图1B)
    3. 双链DNA片段可以购买例如,gBlocks(Integrated DNA Technologies)或GeneStrands(Eurofins Genomics)
  4. 寡核苷酸10pmol /μl
    1. oMS59:5'ATTCGTGATTTACACCAAACACGC 3'
    2. oMS49:5'CCCCTCGTCTCGCGCCTCATTGGTCGAATTG 3'
    3. oSR224:5'CTACACTCAGCACACGATGT 3'
  5. 酶和缓冲液
    1. Acc65I限制性内切核酸酶(New England Biolabs,目录号:R0599S)
    2. Gibson Assembly ®主混合物(New England Biolabs,目录号:E2611S)
    3. Phusion High-Fidelity DNA Polymerase(New England Biolabs,目录号:M0530S)或BioMix Red(Bioline,目录号:BIO-25006)
  6. 套件
    1. QIAprep Spin Miniprep Kit(QIAGEN,目录号:27104)
    2. Wizard SV Gel和PCR Clean-Up系统(Promega,目录号:A9281)
    3. 可选:Plate Seq Kit PCR(Eurofins基因组学)
  7. 抗生素
    1. Milli-Q水中的氨苄西林10 mg / ml原液(Carl Roth,目录号:HP62.1)
    2. Carboxin 5 mg / ml甲醇储备(Sigma-Aldrich,目录号:45371)
  8. 试剂
    1. 胰蛋白胨(BD Biosciences)
    2. 酵母提取物(BD Biosciences)
    3. 氯化钠(NaCl)
    4. 蛋白胨(BD Biosciences)
    5. 蔗糖(BD Biosciences)
    6. 山梨糖醇(Sigma-Aldrich,目录号:S1876)
    7. Bacto琼脂(BD Biosciences)
    8. 马铃薯葡萄糖肉汤(BD Biosciences)
  9. 媒体(见食谱)
    1. 酵母提取物胰蛋白胨(dYT)液体培养基+100μg/ ml氨苄青霉素
    2. YT琼脂平板+100μg/ ml氨苄青霉素
    3. YEPS轻液体培养基+/-2μg/ ml carboxin
    4. 两层再生琼脂灯(RegAgar)板(底层(10毫升),含4微克/毫升羧基和顶层(10毫升),不含羧基(准备新鲜))
    5. 马铃薯葡萄糖(PD)琼脂平板+/-2μg/ ml羧基
    注意:所有介质都需要高压灭菌。对于平板,每个培养皿应使用20毫升培养基。

设备

注意:不需要特定制造商的设备。可以使用任何等效设备。

  1. 移液器
  2. 可上网的电脑
  3. 平板培养箱(28°C,37°C)
  4. 振荡器(28°C,37°C)
  5. ThermoMixer(例如,Eppendorf,型号:ThermoMixer ® C,目录号:5382000015)
  6. 热循环仪(例如,Analytik,Jena,Biometra,目录号:846-2-070-301)
  7. 台式离心机(例如,Thermo Fisher Scientific,目录号:75008801)

软件

不需要特定的软件。可以使用任何适当的对准程序,如CloneManager 9 Professional Edition(科学和教育软件)或CLC Main Workbench(QIAGEN)。

程序

  1. 使用E-CRISP选择目标序列(Heigwer et al。,2014)
    注意:尽管已经为sgRNA设计开发了几种工具,但我们的经验仅限于被证明非常可靠的E-CRISP工具。
    1. 选择 U. maydis 作为E-CRISP网页中的有机体( http://www.e -crisp.org/E-CRISP/ )。
    2. 单击“输入目标序列”框并以FASTA格式插入目标基因组DNA序列。
      注意:使用您想要失活的基因的编码序列并将搜索限制在基因的最5'端。如果您没有找到合适的sgRNA,您可以将输入序列扩展到3'端。
    3. 点击“启动应用程序”面板上的“显示高级选项”,确保3'PAM为NGG。
      注意:
      1. E-CRISP工具提供了将靶序列的搜索限制为以G开头的序列。但是,当使用我们的质粒骨架时,这种限制是不必要的,因为我们的线性化质粒在3'末端保留了G突出端。 U6启动子作为第一个G优先启动U6启动子的转录。
      2. E-CRISP工具为您提供了设计截断目标序列的选项(Fu et al。,2014)。我们通常使用20nt的靶序列,但是使用短至18nt的靶序列已经成功。
    4. 点击“开始sgRNA搜索”。
    5. 选择分数最高且基因中最多5'的靶序列。仔细检查目标基因是否与您想要破坏的基因相对应。
  2. 按照表1中的实例设计sgRNA表达盒并将其插入pCas9_sgRNA_0或pMS73(图1)
    注意:开发用于U. maydis的基因组编辑的CRISPR-Cas9质粒赋予大肠杆菌中的氨苄青霉素抗性和U. maydis中的羧resistance抗性。这些质粒含有用于自主复制的U. maydis ARS元件(Tsukuda等,1988)。质粒主链也在Acc65I位点之前含有U. maydis U6启动子,用于通过Gibson Assembly >(图1)。密码子优化的Cas9的表达由pCas9_sgRNA_0中的组成型otef启动子(图1A)或pMS73中的更强的hsp70启动子驱动(图1B)。


    图1. CRISPR-Cas9质粒的方案和克隆策略。 A.pCas9_sgRNA_0的示意图和用于引入单个sgRNA的克隆策略。可以通过Gibson Assembly ®将用户定义的靶向片段克隆到Acc65I线性化的pCas9_sgRNA_0骨架中。 B.pMS73的示意图和用于引入两个或更多个sgRNA表达盒的克隆策略。用户定义的左右边界盒和可变数量的中间盒可以通过Gibson Assembly ®克隆到Acc65I线性化的pMS73骨架中。靶序列用作Gibson突出端,并且可以根据需要插入的靶序列的数量修改中间盒的数量。 ori:复制起点,bla:β-内酰胺酶基因,赋予 E中氨苄青霉素抗性。大肠杆菌,ARS:在 U中自主复制序列以进行复制。 maydis ,ip R : U. maydis ip allele赋予对carboxin的抗性。黄色框中的数字表示不同的靶向序列。支架序列对应于Cas9结合所需的最小支架(Hsu 等人,,2013)。用于驱动不同sgRNA表达的tRNA启动子由不同颜色的箭头指示。青色线表示Gibson突出到P U6 。绿线表示Gibson突出到P otef 。紫色线表示Gibson突出到P hsp70 。

    1. 在适当的模板中引入您的目标序列(表1)。
    2. 将设计的序列购买为双链DNA片段。
      注意:购买sgRNA表达盒的替代方法是通过模板PCR扩增产生它们。您可以使用双链DNA片段作为此类PCR的模板。

      表1.用于生成含有目标靶序列的双链DNA片段的模板


      表1.续

      *表1中的颜色与图1中的颜色相对应。
      **支架序列对应于Cas9结合所需的最小支架(Hsu et al 。,2013)。

      注意:标记为tRNA启动子的序列包含最终在成熟tRNA中的tRNA的DNA序列和编码成熟tRNA的部分上游100bp。

  3. 通过Gibson Assembly 将sgRNA表达盒克隆到CRISPR质粒中(Gibson et al。,2010)
    1. 用Acc65I线性化pCas9_sgRNA_0或pMS73。
    2. 根据制造商的方案,通过GibsonAssembly®将sgRNA表达盒克隆到骨架中。
      注意,我们使用了Gibson Assembly ® Master Mix(使用手册,NEB#E2611S / L 10/50反应,版本4.0) )。
    3. 将Gibson Assembly ®反应的一部分转化为有能力的 E。根据制造商的方案选择大肠杆菌细胞,并选择在含有100μg/ ml氨苄青霉素的YT平板上生长。将选择板在37°C孵育过夜。
      注意:我们使用了热休克大肠杆菌转化方案(细菌转化工作流程 - 4个主要步骤)在Thermo Fischer网站上给出。
    4. 选择一些菌落以检查sgRNA表达盒的正确插入。使用无菌移液管尖或牙签将单个菌落接种到3 ml含有100μg/ ml氨苄青霉素的dYT培养基中。在37°C振荡孵育过夜。
    5. 根据制造商的说明,使用例如QIAprep spin miniprep试剂盒从细菌培养物中分离质粒DNA。
    6. 通过限制性分析和测序测试具有插入物的质粒。
    注意: 图1. CRISPR-Cas9质粒的方案和克隆策略。 A.pCas9_sgRNA_0的示意图和用于引入单个sgRNA的克隆策略。可以通过Gibson Assembly ®将用户定义的靶向片段克隆到Acc65I线性化的pCas9_sgRNA_0骨架中。 B.pMS73的示意图和用于引入两个或更多个sgRNA表达盒的克隆策略。用户定义的左右边界盒和可变数量的中间盒可以通过Gibson Assembly ®克隆到Acc65I线性化的pMS73骨架中。靶序列用作Gibson突出端,并且可以根据需要插入的靶序列的数量修改中间盒的数量。 ori:复制起点,bla:β-内酰胺酶基因,赋予 E中氨苄青霉素抗性。大肠杆菌,ARS:在 U中自主复制序列以进行复制。 maydis ,ip R : U. maydis ip allele赋予对carboxin的抗性。黄色框中的数字表示不同的靶向序列。支架序列对应于Cas9结合所需的最小支架(Hsu 等人,,2013)。用于驱动不同sgRNA表达的tRNA启动子由不同颜色的箭头指示。青色线表示Gibson突出到P U6 。绿线表示Gibson突出到P otef 。紫色线表示Gibson突出到P hsp70 。

    1. 在适当的模板中引入您的目标序列(表1)。
    2. 将设计的序列购买为双链DNA片段。
      注意:购买sgRNA表达盒的替代方法是通过模板PCR扩增产生它们。您可以使用双链DNA片段作为此类PCR的模板。

      表1.用于生成含有目标靶序列的双链DNA片段的模板


      表1.续

      *表1中的颜色与图1中的颜色相对应。
      **支架序列对应于Cas9结合所需的最小支架(Hsu et al 。,2013)。

      注意:标记为tRNA启动子的序列包含最终在成熟tRNA中的tRNA的DNA序列和编码成熟tRNA的部分上游100bp。
  4. 通过Gibson Assembly 将sgRNA表达盒克隆到CRISPR质粒中(Gibson et al。,2010)
    1. 用Acc65I线性化pCas9_sgRNA_0或pMS73。
    2. 根据制造商的方案,通过GibsonAssembly®将sgRNA表达盒克隆到骨架中。
      注意,我们使用了Gibson Assembly ® Master Mix(使用手册,NEB#E2611S / L 10/50反应,版本4.0) )。
    3. 将Gibson Assembly ®反应的一部分转化为有能力的 E。根据制造商的方案选择大肠杆菌细胞,并选择在含有100μg/ ml氨苄青霉素的YT平板上生长。将选择板在37°C孵育过夜。
      注意:我们使用了热休克大肠杆菌转化方案(细菌转化工作流程 - 4个主要步骤)在Thermo Fischer网站上给出。
    4. 选择一些菌落以检查sgRNA表达盒的正确插入。使用无菌移液管尖或牙签将单个菌落接种到3 ml含有100μg/ ml氨苄青霉素的dYT培养基中。在37°C振荡孵育过夜。
    5. 根据制造商的说明,使用例如QIAprep spin miniprep试剂盒从细菌培养物中分离质粒DNA。
    6. 通过限制性分析和测序测试具有插入物的质粒。
    注意:...
  5. 在此步骤中,您可以通过将PD-carboxin平板上的单个菌落平行复制平板来确认CRISPR-Cas9质粒的丢失(参见步骤8)以产生主板并从主板中选择那些菌落。不能在carboxin板上生长的盘子。


    图2.含有要测试的菌落的主板的实例。建议生成主板以促进筛选过程。单个菌落来自个体 U.将maydis 转化体按照网格图案转移到新的PD板上,并用数字标记以进行鉴定。用扁平牙签进行转移,将细胞置于网格正方形的中心并稍微扩展以增加细胞可以长大并形成菌落的区域。 A.孵化前的主板; B.在28℃温育过夜后制成平板。

  • 基于菌落PCR的筛选用于CRISPR-Cas9诱导的突变
    1. 设计引物,能够扩增含有靶位点的约500bp的PCR产物。
    2. 使用无菌移液管尖或牙签,从主板上取少量菌落(步骤6c),并将其置于含有20μl20mM NaOH的96孔板的孔中。
    3. 使用例如(ThermoMixer)在室温下以800rpm摇动板30分钟。
    4. 使用高保真聚合酶例如,Phusion DNA聚合酶或BioMix Red建立20μlPCR反应,如制造商所建议的。使用1μl裂解细胞作为DNA模板。
      注意:在多路复用的情况下,需要对每个目标位点进行单独的PCR。如果您有许多菌落和/或位点需要测试,我们建议您使用Plate Seq Kit PCR等试剂盒。在这种情况下,在该步骤中直接在试剂盒中提供的96孔PCR板中进行PCR反应。
    5. 按照聚合酶制造商的说明,在Thermocycler中扩增PCR产物。
    6. 在用Eurofins的Plate Seq试剂盒中用例如,Wizard SV凝胶和PCR净化系统或例如清除之后对PCR片段进行测序。
  • CRISPR-Cas9质粒缺失
    1. 如果您之前没有筛选质粒的丢失(参见步骤6之后的注释),用主板上的牙签挑选一些细胞,并在含有羧基的PD平板上划线。在28°C孵育两天。回到主板并使用不能在carboxin板上生长的菌落,这些菌落丢失了质粒。
      注意:我们观察到质粒在主板上的菌落中保留了约5%的病例。
    2. 在carboxin平板上的生长意味着一些细胞仍然含有质粒。如果是这种情况,并且该菌落含有所需的突变,则在3ml不含羧基的YESPSL培养基中接种来自该菌株的细胞,并在28°C下生长过夜。
    3. 从这种过夜培养物中取出细胞并铺在不含羧基的PD平板上以获得单菌落。在28°C孵育两天。
    4. 两者上的复制板单菌落,具有羧基的PD板和不含羧基的PD板,并在28℃下孵育过夜。选择丢失质粒的菌落即,无法在含有羧基的平板上生长。
    5. 如有必要,重复质粒程序的丢失,直到选择平板上没有生长。
    注意:重新排序最终克隆以验证您是否已获得所需的突变。
  • 数据分析

    应使用CloneManager或CLC Main Workbench等软件通过与野生型序列比对来分析测序数据。当缺失或插入的核苷酸数量不能除以3时,产生移码突变。或者,可以使用产生过早终止密码子的插入缺失。必须仔细检查测序色谱图并检查是否存在重叠峰。重叠峰是序列样品中遗传异质性的标志(图3)。


    图3.代表性测序结果。野生型(WT)等位基因的核苷酸序列和色谱图的比对,缺失19bp的相应编辑等位基因和来自相同实验的异源样品。注意,测序公司为异源样品提供了一个序列(深橙色),考虑到最高峰并错误地提示序列同质性。只有通过仔细分析色谱图才能识别异质性,在这种情况下,色谱图显示直至箭头的序列(预测的Cas9切割位点)是均一的,并且从这一点开始变得异质。靶序列和PAM序列在野生型序列中描绘为彩色框。使用CLC Main Workbench生成对齐。

    笔记

    1. U中的CRISPR-Cas9系统。已证明maydis 对单基因的破坏是高效的,其中高达100%的测试转化体含有编辑的细胞(Schuster 等人,,2016)。对于编辑效率较低的sgRNA,使用更强的启动子( ie ,P hsp70 而不是P otef <更高表达Cas9蛋白可以提高效率)通过在选择Cas9表达质粒的条件下将转化的细胞保持在液体培养物中来延长对Cas9的暴露(Schuster 等,,2018)。使用更长的Cas9曝光时间,我们已经能够使用多种sgRNA改进编辑。我们很少遇到目标序列,它们根本不起作用或者基因编辑效率很低。在这种情况下,改变目标序列提供了有效的解决方案。
    2. 已经分析了两个独立编辑的菌株的脱靶突变,并且没有通过全基因组测序发现的脱靶(Schuster et al。,2016)。由于脱靶对每个sgRNA都是特异性的,我们建议在给定基因或基因组中产生三个独立的突变体。如果这些突变体都表现出相同的表型改变,并且这种表型可以通过引入野生型基因来补充,那么您就有令人信服的证据表明该表型是由产生的突变引起的。
    3. 在设计多路复用实验时,应考虑待编辑基因的基因组位置。同一染色体中的两个或更多个同时的CRISPR事件可能导致不希望的染色体缺失或重排。在这种情况下,应该执行顺序编辑。

    食谱

    1. 酵母提取物胰蛋白胨(dYT)培养基
      该媒介包括:
      1.6%(w / v)胰蛋白胨(BD Biosciences)
      1%(w / v)酵母提取物(BD Biosciences)
      双蒸水中0.5%(w / v)NaCl
      注意:高压灭菌并在使用前储存在室温下。
    2. 酵母提取物胰蛋白胨(YT)琼脂培养基
      该媒介包括:
      0.8%(w / v)胰蛋白胨(BD Biosciences)
      0.5%(w / v)酵母提取物(BD Biosciences)
      0.5%(w / v)NaCl
      双蒸水中1.3%Bacto琼脂(BD Biosciences)
      注意:高压灭菌并立即倒入培养皿(每个培养皿20毫升)。
    3. YEPSL液体培养基
      该媒介包括:
      0.4%(w / v)酵母提取物(BD Biosciences)
      0.4%(w / v)蛋白胨(BD Biosciences)
      双蒸水中2%(w / v)蔗糖(BD Biosciences)
      注意:高压灭菌并在使用前储存在室温下。
    4. 再生琼脂灯(RegAgar)
      该媒介包括:
      1%(w / v)酵母提取物(BD Biosciences)
      0.4%(w / v)蛋白胨(BD Biosciences)
      0.4%(w / v)蔗糖(罗斯)
      18.22%(w / v)山梨糖醇(Sigma)
      双蒸水中1.5%Bacto琼脂(BD Biosciences)
      注意:
      1. 高压灭菌并在使用前储存在室温下。
      2. 在U. maydis转化期间,将琼脂在微波炉中熔化,充分混合并冷却至50-60℃。加入Carboxin(4μg/ ml终浓度),将10ml该补充的RegAgar倒入培养皿中。一旦固化,将另外10ml不含羧基的熔融RegAgar作为顶层倒入相同的培养皿中。在铺设转化混合物之前,应将该第二层倒入约25-30分钟(参见Bösch等人,2016年的转化方案)。&nbsp;
    5. 马铃薯葡萄糖(PD)琼脂培养基
      该媒介包括:
      2.4%马铃薯葡萄糖肉汤(BD Biosciences)
      2%Bacto琼脂(BD Biosciences)在双蒸水中
      注意:高压灭菌并立即倒入培养皿(每个培养皿20毫升)。

    致谢

    这项工作由马克斯普朗克协会资助。该协议改编自Schuster 等人(2016年和2018年)。我们要感谢Kahmann实验室的现任和前任成员,他们定期使用CRISPR-Cas9介导的基因组编辑,并提供有关该方案的反馈。作者声明,没有与本文相关的利益冲突或竞争利益。

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    引用:Schuster, M., Trippel, C., Happel, P., Lanver, D., Reißmann, S. and Kahmann, R. (2018). Single and Multiplexed Gene Editing in Ustilago maydis Using CRISPR-Cas9. Bio-protocol 8(14): e2928. DOI: 10.21769/BioProtoc.2928.
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