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A Golden Gate-based Protocol for Assembly of Multiplexed gRNA Expression Arrays for CRISPR/Cas9
基于Golden Gate法的CRISPR/Cas9多重gRNA表达阵列组装方案   

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Abstract

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) has become the most broadly used and powerful tool for genome editing. Many applications of CRISPR-Cas9 require the delivery of multiple small guide RNAs (gRNAs) into the same cell in order to achieve multiplexed gene editing or regulation. Using traditional co-transfection of single gRNA expression vectors, the likelihood of delivering several gRNAs into the same cell decreases in accordance with the number of gRNAs. Thus, we have developed a method to efficiently assemble gRNA expression cassettes (2-30 gRNAs) into one single vector using a Golden-Gate assembly method (Vad-Nielsen et al., 2016). In this protocol, we describe the detailed step-by-step instructions for assembly of the multiplexed gRNA expression array. The gRNA scaffold used in our expression array is the gRNA 1.0 system for the Cas9 protein from Streptococcus pyogenes driven by the human U6 promoter.

Keywords: CRISPR(CRISPR), SpCas9(SpCas9), Golden-Gate assembly(Golden-Gate组装), Multiplexed gRNA array(多重gRNA阵列), Simultaneously genetic manipulation(同步遗传操纵)

Background

The broadened CRISPR toolbox based on wild-type Cas9 or nuclease-deficient Cas9 (dCas9) has greatly facilitated genome/epigenome editing and regulation in all organisms. Multiplexed gene editing or regulation requires simultaneous expression of several gRNAs in the same cell. The traditional way of delivering several gRNAs into cells is based on either co-transfection of individual gRNA expression vectors or generation of a vector carrying multiple gRNA expression cassettes using traditional cloning; a process which is extremely time consuming. Another way of generating a vector containing multiple gRNA expression cassettes is based on gene synthesis, which is costly and only applicable when working with a very limited number of gRNA expression cassettes. The current protocol is based on Golden Gate cloning which can be used to assemble up to 30 individual gRNA expression cassettes into a single vector within 7 days (Figure 1), with each cassette being driven by an individual human U6 promoter. In our study, we have validated the applicability of this system in both human and porcine cells, but it is in principle compatible with applications in any other organisms that can utilize the human U6 promoter. Compared with existing methods, our method is cost effective, rapid (7 days) and flexible (applicable with any gRNAs that do not contain a BbsI, BsaI or BsmBI recognition site). Many applications of CRISPR/Cas9 may benefit from using our system, including multiplexed gene knockout by CRISPR/SpCas9, multiplexed gene inhibition by CRISPRi, and multiplexed gene activation by CRISPRa.


Figure 1. Schematic illustration of the principle of the current protocol. The current protocol is carried out in 2-3 major steps which vary depending on the number of gRNA expression cassettes to be assembled. Step 1: The gRNA oligonucleotides (T#) are cloned into individual modular single gRNA expression vectors (pMA-SpCas9-g#). Step 2: The individual gRNA expression vectors (pMA-T#) are assembled into 1-3 array vectors depending on the total number of gRNAs. Step 3: For assembly of 11-30 gRNA expression cassettes, 2 to 3 individual array vectors are subjected to a second round of assembly to yield the final EGFP expressing vector (pMsgRNA-EGFP).

Materials and Reagents

  1. 200 µl PCR tubes
  2. 1.5 ml Eppendorf tubes
  3. 10 or 100 µl pipette tips
  4. Competent E. coli cells (No particular preference, but should be recombination deficient)
  5. The modular gRNA plasmids (available from Addgene, see Table 1 for corresponding Addgene plasmid IDs)
  6. The pFUS-B1 to pFUS-B10, pFUS-A, pFUS-A30A and pFUS-A30B plasmids (available from Addgene, Golden Gate TALEN and TAL Effector Kit 2.0) (Addgene, catalog number: 1000000024 )
  7. NEB buffer 2 (New England BioLabs, catalog number: B7002S )
  8. BbsI (FastDigest) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD1014 )
  9. T4 DNA ligase (5 U/μl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EL0014 )
  10. Distilled H2O
  11. Ampicillin (Sigma-Aldrich, catalog number: A1593 )
  12. LB medium
  13. dNTP
  14. DreamTaq or other equivalent DNA polymerase (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: K1072 )
  15. Agarose
  16. Plasmid prep mini kit (No particular preference, we used the Nucleo Spin plasmid easy pure kit from MACHEREY-NAGEL, catalog number: 740727 )
  17. BsaI (BpiI) (FastDigest) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0293 )
  18. Plasmid-SafeTM ATP-Dependent DNase (Epicentre, catalog number: E3101K , ATP is included in this kit)
  19. Spectinomycin (Sigma-Aldrich, catalog number: PHR1441 )
  20. X-gal (Sigma-Aldrich, catalog number: B4252 )
  21. IPTG (dissolved IPTG in water) (Sigma-Aldrich, catalog number: I6758 )
  22. AflII (BspTI) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0834 )
  23. XbaI (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0684 )
  24. BsmBI (Esp3I) (FastDigest) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0454 )
  25. Universal primers for PCR screening (see Table 1)

    Table 1. Plasmids and primers. Plasmids developed for this protocol are available from Addgene with corresponding Addgene IDs. Single modular plasmids (pMA-SpCas9-g1 to pMA-SpCas9-10) for cloning gRNA oligonucleotides into individual gRNA expression plasmids. Array plasmid (pMA-MsgRNA-EGFP) for assembly of gRNA expression array containing 11-30 gRNA expression cassettes. Primer sequences used in this protocol for screening of assembled array plasmids.
    Plasmid
    Addgene ID
    pMA-MsgRNA-EGFP
    80794
    pMA-SpCas9-g10
    80793
    pMA-SpCas9-g9
    80792
    pMA-SpCas9-g8
    80791
    pMA-SpCas9-g7
    80790
    pMA-SpCas9-g6
    80789
    pMA-SpCas9-g5
    80788
    pMA-SpCas9-g4
    80787
    pMA-SpCas9-g3
    80786
    pMA-SpCas9-g2
    80785
    pMA-SpCas9-g1
    80784
    Primers
    Sequences (5’-)
    Universal U6 Forward
    ATAAGGATCCGGTCTCGCTATGAGGGCCTATTTCCCATG
    Universal Scr Reverse
    ATAATGTACAGGTCTCCCATGTAACTTGCTATTTCTAGCTC
    Note: The Universal U6 and Scr primers have been modified to suit the PCR conditions in this protocol.

Equipment

  1. Heating block (Grant Instruments, model: QBD2 )
  2. Microcentrifuge (Eppendorf, model: Centrifuge 5424 )
  3. 37 °C Thermo incubator (Labnet International, model: 211DS )
  4. 37 °C shaking incubator (environmental incubator shaker G24 ) (Eppendorf, New Brunswick Scientific, model: G24)
  5. Thermal cycler (Thermo Fisher Scientific, Applied BiosystemsTM, model: Veriti® 96 well thermal cycler )
  6. DNA electrophoresis apparatus (Bio-Rad Laboratories, model: Wide mini-sub cell GT )
  7. Nanodrop (Thermo Fisher Scientific, model: Nanodrop 1000 Spectrophotometer )

Procedure

  1. Preparation: design and order CRISPR gRNA oligos
    1. Design target gRNA oligonucleotides using any of the online designing tools such as the optimized CRISPR design tool (http://crispr.mit.edu/).
      Notes:
      1. Because the gRNA expression cassette utilizes a human U6 promoter, the first nucleotide of the CRISPR target site should preferably be a ‘G’ to ensure efficient transcription.
      2. It is also very important to exclude gRNA oligonucleotides that contain any of the following type IIS restriction sites: BbsI, BsaI, and BsmBI.
    2. For target sites starting with a ‘G’, add the following four nucleotide overhangs to the sense (SS) and antisense (AS) gRNA oligonucleotides:
      SS: 5’CACC(N20)
      AS: 5’AAAC(N20)
    3. For target sites not stating with a ‘G’, add the following overhangs:
      SS: 5’CACCG(N20)
      AS: 5’AAAC(N20)C
    4. Order oligonucleotides from any qualified company, dilute the oligonucleotides to a stock concentration of 100 pmol/µl (µM) and store at -20 °C. (Standard desalted purification of the oligos is sufficient).

  2. Annealing of gRNA oligos
    1. Anneal the sense and antisense gRNA oligonucleotides for each gRNA in separate 1.5 ml EP tubes:
      Sense gRNA oligo: 1 µl (100 pmol/µl)
      Antisense gRNA oligo: 1 µl (100 pmol/µl)
      10x NEB buffer 2: 2 µl
      Add ddH2O to a final volume of 20 µl.
    2. Denature the mixture at 95 °C for 5 min in a heating block.
    3. Place the heating block on the lab bench and slowly anneal oligonucleotides until mixture is at room temperature (which takes approximately 1-2 h).
    4. Quick-spin the annealed gRNA oligonucleotides to the bottom of the tubes and store at -20 °C for later use or proceed with the following experimental procedure.

  3. Vector cloning
    Using this protocol, the generation of the gRNA expression array vector is accomplished in 5 days (for 1 to 10 gRNA expression cassettes) to 7 days (for 11 to 30 cassettes). See schematic in Figure 2 for an overview. In the protocol, the location of a target site in the CRISPR expression array is referred to as T# (#, an integer between 1 and 30 denoting the order of the gRNAs). The protocol utilizes 10 individual gRNA expression cassettes that are tandemly assembled into a set of ‘array’ vectors depending on the number of cassettes, where each array vector contains up to 10 cassettes. When assembling 11-30 cassettes, multiple array vectors are finally assembled into a single vector. The selection of plasmids for assembly is described in step C3c, Table 3. A schematic illustration is given in Figure 1.


    Figure 2. Protocol overview. Day to day overview of the protocol. For assembly 1 to 10 gRNA expression cassettes, the protocol is completed after miniprep of positive colonies at day 5. For assembling 11 to 30 gRNA expression cassettes, an additional round of assembly is performed at day 5 and completed after day 7.

    1. Day 1. Ligation of gRNA oligonucleotides into individual modular single gRNA expression vectors
      1. Select pMA plasmids for annealed gRNA oligonucleotides T1-TN (N ≤ 30) generated in step B4 according to the table below (pMA-SpCas9-g1 for T1, pMA-SpCas9-g2 for T2, …, pMA-SpCas9-g1 for T11, pMA-SpCas9-g2 for T12, …, pMA-SpCas9-g1 for 21, pMA-SpCas9-g2 for T22, etc.) (Table 2).

        Table 2. Assembly scheme of modular single gRNA expression vectors and annealed gRNA oligonucleotides. This table illustrates how to choose the right single modular plasmids (pMA-SpCas9-g1 to pMA-SpCas9-10) for cloning of the gRNA oligonucleotides into individual gRNA expression plasmids (named pMA-T# after successful cloning). The number of each gRNA represents the position of the gRNA in the tandem gRNA expression array.


      2. Prepare assembly reaction in individual PCR tubes containing:
        1. 100 ng modular single gRNA expression plasmid (pMA-SpCas9-g#).
        2. 1 µl of annealed gRNA oligonucleotide from step B4 (T#).
        3. 1 µl BbsI restriction enzyme.
        4. 1 µl T4 DNA ligase.
        5. 2 µl 10x T4 ligase buffer (to a final concentration of 1x).
        6. Nuclease-free water up to 20 µl total reaction volume.
      3. Incubate the reactions in a thermal cycler with the following conditions:


      4. Transform competent E. coli using 2 µl of the ligation product.
        Note: Protocols for generation of competent E. coli and transformation are available from the authors upon request.
      5. Plate (1/10) transformed cells on a 10 cm LB agar plate containing 50 µg/ml ampicillin.
        Note: Do not over-plate the cells.
      6. Incubate agar plates at 37 °C overnight.
    2. Day 2. 1st colony screening and culture
      1. Pick two to three colonies from each transformation using a 10 or 100 µl pipette tip.
      2. Dip the pipette tip in 100 µl LB medium (50 µg/ml ampicillin) to start initial culture. Transfer the pipette tip to a PCR tube containing 30 µl nuclease-free water and pipette up and down to release cells.
        Note: The initial culture and PCR tube should be labeled with the same ID. If working with over 10 gRNAs, we normally use two sets of 200 µl PCR tubes, one set for the initial culture and the other set for the cell lysate.
      3. Place the initial culture tubes in a 37 °C incubator.
      4. Place the PCR tubes in a thermal cycler at 98 °C for 10 min to lyse cells.
      5. Perform gRNA PCR screenings of cell lysates. Add to each reaction:
        1. 1 µl transformed E. coli lysate.
        2. 5 pmol universal U6 Forward primer (Table 1).
        3. 5 pmol antisense oligonucleotide of the gRNA in question.
        4. 0.4 µl dNTP (10 mM).
        5. 0.1 µl DreamTaq polymerase (or similar).
        6. 2 µl 10x DreamTaq buffer (to final concentration of 1x).
        7. Nuclease-free water to 20 µl total reaction volume.
        Example: For colony screening of pMA-SpCas9-T1, PCR is performed for 2-3 picked colonies using the universal U6 promoter as the forward primer and T1-antisense oligonucleotide as the reverse primer.
      6. Incubate the PCR reactions in a thermal cycler with the following conditions:


      7. Visualize PCR product on a 1% agarose gel by electrophoresis. The expected amplicon size is approximately 270 bp.
      8. Select one positive colony from each transformation and transfer 50 µl of initial culture to individual tubes containing 5 ml LB medium with 50 µg/ml ampicillin.
      9. Incubate overnight at 37 °C in an orbital shaker.
    3. Day 3. Plasmid prep and assembly of individual gRNA expression cassettes into the ‘array’ plasmids
      1. Isolate the plasmid DNA (named pMA-T#) from the overnight cultures using a commercial miniprep kit according to the manufacturer’s instructions.
      2. Validate the single gRNA expression plasmids by Sanger sequencing using the universal U6 primer (recommended).
      3. Select array plasmids for 1st assembly of single gRNA expression plasmids according to Table 3:

        Table 3. Selection of array plasmids for assembly of pMA-T# vectors. Selection of array plasmids depends on the number of gRNA expression cassettes to be assembled. For assembly of 1-10 gRNA cassettes (Left), one of the pFUS-B# array plasmids is selected depending on the number of gRNA cassettes. When assembling 11-20 gRNA cassettes (Middle), pFUS-A is selected for the assembly of gRNA cassettes 1 to 10. For the remaining cassettes (≤ 10), one of the pFUS-B# array plasmids is selected depending on the number of gRNA cassettes. For assembly of 21-30 gRNA cassettes (Right), pFUS-A30A is used for assembly of the first 10 gRNA expression cassettes. pFUS-A30B is used for assembling gRNA cassettes 11 to 20. The remaining cassettes (≤ 10) are cloned into a pFUS-B# array plasmid depending on the number of gRNA cassettes.


      4. Set up Golden Gate assembly reactions in individual PCR tubes containing:
        1. 30 ng array plasmid.
          Note: The plasmid amount has been optimized.
        2. 50 ng of each individual modular single gRNA plasmid (maximum of 10 plasmids).
        3. 1 µl BsaI restriction enzyme.
        4. 1 µl T4 DNA ligase.
        5. 2 µl 10x T4 ligase buffer (to a final concentration of 1x).
        6. Nuclease-free water to 20 µl total reaction volume.
      5. Incubate the reactions in a thermal cycler with the following conditions:


      6. Add 1 µl 25 mM ATP and 1 µl plasmid safe DNase to the reaction.
        Note: Plasmid safe DNase treatment digests all unligated linear dsDNA fragments, incomplete assembly products and linearized vectors. This step minimizes the risk of in vivo recombination of linearized dsDNA fragments in the competent cell. This step is very important when assembling more than five gRNA expression cassettes.
      7. Incubate the reactions in a thermal cycler for 1 h at 37 °C.
      8. Transform competent E. coli cells using 2 µl of the assembled product.
      9. Plate (1/20) of the transformed cells on 10 cm LB agar plates containing 50 µg/ml spectinomycin as well as 8 µl 100 mg/µl X-gal and 8 µl 0.5 M IPTG for blue/white colony screening.
      10. Incubate agar plates at 37 °C overnight.
    4. Day 4. 2nd colony screening and culture
      1. Inspect plates for positive white colonies. The expected ratio of white to blue colonies is at least 25% for successful assembly of 10 gRNA expression cassettes and will increase gradually with a decreased number of gRNA expression cassettes (Figure 3).


        Figure 3. Inspection and screening of first round of assembling. Left: The transformation plate for M10. White colonies contain vectors with insertions. Blue colonies contain empty vectors. Right: Universal PCR for M10 screening of 8 white colonies. Lanes 1-4 and 7-8 show multiple bands resembling a ladder indicative of a positive colony. Lanes 5 and 6 only show the two dominant bands without a ladder indicative of an incorrect insertion.

      2. Pick two to three white colonies from each transformation as described for Day 2.
      3. Set up PCR screening reactions for all picked colonies in individual PCR tubes containing:
        1. 1 µl transformed E. coli lysate.
        2. 5 pmol universal U6 Forward primer (Table 1).
        3. 5 pmol universal Scr Reverse primer (Table 1).
        4. 0.4 µl dNTP.
        5. 0.1 µl DreamTaq polymerase (or similar).
        6. 2 µl 10x DreamTaq buffer (to a final concentration of 1x).
        7. Nuclease-free water to 20 µl total reaction volume.
      4. Incubate the PCR reactions in a thermal cycler with the following conditions:


      5. Visualize PCR product on a 1% agarose gel by electrophoresis. The correct amplification pattern should be a ‘ladder’ with a dominant band at approximately 400 bp and an increase of 392 bp for each ‘step’ corresponding to each additional gRNA expression cassette included in the amplification (Figure 3B).
      6. Select one positive colony from each transformation and transfer 50 µl of initial culture to individual tubes containing 5 ml LB medium with 50 µg/ml spectinomycin.
      7. Incubate overnight at 37 °C in an orbital shaker.
    5. Day 5. Plasmid DNA prep and 2nd assembly for plasmids containing 11 to 30 expression cassettes
      1. Isolate the plasmid DNA from the overnight cultures using a commercial miniprep kit according to the manufacturer’s instructions.
      2. Validate the assembled array plasmids by restriction enzyme digestion. In a 1.5 ml EP tube, add:
        1. 0.5 µg assembled array plasmid DNA.
        2. 1 µl AflII restriction enzyme.
        3. 1 µl XbaI restriction enzyme.
        4. 2 µl 10x reaction buffer (to a final concentration of 1x).
        5. Nuclease-free water to 20 µl total reaction volume.
      3. Visualize digestion product on a 1% agarose gel by electrophoresis.
        Note: The expected size of the released expression cassette is approximately 4 kb for 10 cassettes (~400 bp per gRNA expression cassette). We strongly recommend generating a theoretical sequence map for each multiplex vector. The RFLP results will be different if there are AflII or Xbal recognition sites in any of the gRNA guide sequences.
      4. For further validation of the assembled array plasmid, set up guide specific PCRs for all gRNA expression cassettes present in the array vector in individual PCR tubes.
        Add to each PCR reaction:
        1. 1 ng array plasmid DNA.
        2. 5 pmol sense oligonucleotide of the specific gRNA in pMA-TN as the forward primer.
        3. 5 pmol antisense oligonucleotide of the next specific gRNA in pMA-TN+1 as the reverse primer.
        4. 0.4 µl dNTP.
        5. 0.1 µl DreamTaq polymerase (or similar).
        6. 2 µl 10x DreamTaq buffer (to a final concentration of 1x).
        7. Nuclease-free water to 20 µl total reaction volume.
        Example: For validation of assembled pFUS-B4-M4, three individual PCR reactions should be performed. The first one is with the SS oligonucleotide of T1 as the forward primer and the AS oligonucleotide of T2 as the reverse primer. The second one is with the SS oligonucleotide of T2 as the forward primer and the AS oligonucleotide of T3 as the reverse primer. The third one is with the SS oligonucleotide of T3 as the forward primer and the AS oligonucleotide of T4 as the reverse primer. The total number of PCR reactions per assembled array plasmid is N-1 (N is the number of gRNA expression cassettes contained in the array plasmid) (Figure 4).
      5. Incubate the PCR reactions in a thermal cycler with the following conditions:


      6. Visualize PCR product on a 1% agarose gel by electrophoresis. The expected amplicon sizes are approximately 400 bp.


        Figure 4. Guide specific PCR validation. Top: Schematic illustration of primers used for guide specific PCR. Bottom: An example of a guide specific PCR using a pFUS-B4-M4 plasmid visualized on a 1% agarose gel.

      7. Set up 2nd assembly of array plasmids in a PCR tube (only for assembly of 11-30 cassettes) containing:
        1. 50 ng DNA of each assembled array vector (pFUS-A-M10 + pFUS-B#-M# OR pFUS-A30A-M10 + pFUS-A30B-M10 + pFUS-B#-M#).
        2. 50 ng DNA of array plasmid pMsgRNA-EGFP (Table 1).
        3. 1 µl BsmBI restriction enzyme.
        4. 1 µl T4 DNA ligase.
        5. 2 µl 10x T4 ligase buffer (to a final concentration of 1x).
        6. Nuclease-free water to 20 µl total reaction volume.
      8. Incubate the reaction in a thermal cycler with the following conditions:


      9. Transform competent E. coli cells using 2 µl of the ligation product.
      10. Plate (1/10) of the transformed cells on a 10 cm LB agar plate containing 50 µg/ml ampicillin as well as 40 μl of 20 mg/ml X-gal and 40 μl of 0.1 M IPTG for blue/white colony screening.
      11. Incubate agar plate at 37 °C overnight.
    6. Day 6. 3rd colony screening and culture
      1. Inspect plate for positive white colonies. The expected ratio of white to blue colonies is > 80% for successful assembly.
      2. Pick two to three white colonies as described for Day 2.
      3. Set up overlap PCR screening reactions for each assembled section of gRNA expression cassettes for all picked colonies. To individual PCR tubes, add:
        1. 1 µl transformed E. coli lysate.
        2. 5 pmol sense oligonucleotide of the last gRNA (T10 or T20) in the first or second expression cassette array as the forward primer.
        3. 5 pmol antisense oligonucleotide of the first gRNA (T11 or T21) in the second or third expression cassette array as the reverse primer.
        4. 0.4 µl dNTP.
        5. 0.1 µl DreamTaq polymerase (or similar).
        6. 2 µl 10x DreamTaq buffer (to a final concentration of 1x).
        7. Nuclease-free water to 20 µl total reaction volume.
        Example: For validation of assembled pMsgRNA-EGFP-M30 (array plasmid containing 30 gRNA expression cassettes generated from the assembly of pFUS-A30A – pFUS-A30B – pFUS-B10), two individual overlap PCR reactions are performed. The first reaction uses the sense (SS) oligonucleotide of T10 as the forward primer and the antisense (AS) oligonucleotide of T11 as the reverse primer. The second uses the SS oligonucleotide of T20 as the forward primer and the AS oligonucleotide of T21 (Figure 5).
      4. Incubate the PCR reactions in a thermal cycler with the following conditions:


      5. Visualize PCR product on a 1% agarose gel by electrophoresis. The expected amplicon size is approximately 460 bp.
      6. (Optional) Perform overlap PCRs for all neighboring gRNAs.


        Figure 5. Overlap PCR screening. Top: Schematic illustration of overlap PCR screening of pMsgRNA-EGFP-M30. Bottom: Example of guide specific PCR validation of pMsgRNA-EGFP-M30 visualized on a 1% agarose gel. Lane 10 and lane 20 contain the overlap PCR reactions described above. Note that the overlap PCR produces a slightly larger band than the guide specific PCRs. Generating a theoretical plasmid sequence map is highly recommended as a guideline for assembly, PCR validation, and restriction enzyme digestions.

      7. (Optional) Sanger sequencing using T# as primers.
      8. Select one positive colony and transfer 50 µl of initial culture to individual tubes containing 5 ml LB medium with 50 µg/ml ampicillin added.
      9. Incubate overnight at 37 °C in an orbital shaker.
    7. Day 7. Plasmid DNA preparation and validation
      1. Isolate the plasmid DNA from the overnight culture using a commercial miniprep kit according to manufacturer’s instructions.
      2. Validate the assembled pMsgRNA-EGFP-M# array plasmid by restriction enzyme digestion to release the combined gRNA expression array cassette. Add to a PCR tube:
        1. 0.5 µg assembled array plasmid DNA.
        2. 1 µl SfiI restriction enzyme.
        3. 2 µl 10x reaction buffer (to a final concentration of 1x).
        4. Nuclease-free water to 20 µl total reaction volume.
      3. Visualize digestion product on a 1% agarose gel by electrophoresis. The expected size of the released expression cassette is approximately 1662 + N x 379 bp (N is the total number of gRNA expression cassettes contained in the final array plasmid).

Notes

  1. For the efficiency of cloning in each assembly reaction, we generally experience higher than 95% positive rates for the single gRNA expression vectors. This positive rate is based on PCR screening. The positive rate for the assembly of gRNA expression arrays is based on the ratio of white (positive) to blue (negative) colonies. Please note that all blue colonies are negative. However, the white colonies may also be negative if they contain incorrect ligation or recombination products. It is therefore important to perform PCR screenings.
  2. If a high blue colony rate is observed, it is recommended to check the expiry date of your enzymes as the FastDigest enzymes have a relatively short storage time. Other measures such as lowering the amount of the pFUS plasmid in assembly reactions, increasing cycle numbers, and increasing the amount of enzyme can increase the white colony rate as well.

Acknowledgments

JVN and ALN were supported by grants from the Lundbeck Foundation, Krista og Viggo Petersens Fond, Fabrikant Einar Willumsens Mindelegat, and Fonden til Lægevidenskabens Fremme. LL and LB were supported by the DREAM project from the Lundbeck Foundation. YL was supported by grants from the Danish Research Council for Independent Research, the Sapere Aude Young Research Talent prize to YL, the Lundbeck Foundation and the Innovation Fund Denmark (BrainStem). This protocol is developed based on our previous study published in Cell Mol Life Sci (Vad-Nielsen et al., 2016). We also acknowledge Voytas DF’s group, who constructed plasmids for the first assembly used in this protocol (Cermak et al., 2011).

References

  1. Cermak, T., Doyle, E. L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., Baller, J. A., Somia, N. V., Bogdanove, A. J. and Voytas, D. F. (2011). Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39(12): e82.
  2. Vad-Nielsen, J., Lin, L., Bolund, L., Nielsen, A. L. and Luo, Y. (2016). Golden Gate Assembly of CRISPR gRNA expression array for simultaneously targeting multiple genes. Cell Mol Life Sci 73(22): 4315-4325.

简介

CRISPR(聚簇定期间隔短回文重复序列)相关蛋白9(Cas9)已成为最广泛使用和强大的基因组编辑工具。 CRISPR-Cas9的许多应用需要将多个小导向RNA(gRNA)递送到相同的细胞中以实现多重基因编辑或调节。使用单个gRNA表达载体的传统共转染,将几个gRNA递送到相同细胞中的可能性根据gRNA的数量减少。因此,我们开发了使用Golden-Gate装配方法(Vad-Nielsen等人,2016)将gRNA表达盒(2-30gRNA)有效地装配到一个单一载体中的方法。在这个协议,我们描述详细的分步指示的多路复用gRNA表达式数组的组装。在我们的表达阵列中使用的gRNA支架是由人U6启动子驱动的来自化脓性链球菌的Cas9蛋白的gRNA 1.0系统。
关键字: CRISPR,SpCas9,金门汇编,多重gRNA阵列,同时遗传操作 

[背景] 基于野生型Cas9或核酸酶缺陷型Cas9(dCas9)的CRISPR工具箱大大促进了所有生物体中的基因组/表观基因组编辑和调控。多重基因编辑或调节需要在同一细胞中同时表达几种gRNA。将几种gRNA递送到细胞中的传统方法是基于使用传统克隆共转染个体gRNA表达载体或产生携带多个gRNA表达盒的载体;这是非常耗时的过程。产生含有多个gRNA表达盒的载体的另一种方法是基于基因合成,其是昂贵的并且仅在使用非常有限数量的gRNA表达盒时才适用。目前的方案基于金门克隆,其可用于在7天内将多达30个个体gRNA表达盒装配成单个载体(图1),其中每个盒由单个人U6启动子驱动。在我们的研究中,我们已经验证了该系统在人和猪细胞中的适用性,但它原则上与在可以利用人类U6启动子的任何其他生物体中的应用相容。与现有方法相比,我们的方法是成本有效的,快速的(7天)和灵活的(适用于不含有Bbs I, Bsa I或 > BsmB I识别位点)。 CRISPR/Cas9的许多应用可能受益于使用我们的系统,包括通过CRISPR/SpCas9的多重基因敲除,CRISPRi的多重基因抑制和CRISPRa的多重基因激活。



图1.当前协议原理示意图。 目前的协议是在2-3个主要步骤中进行的,这取决于要组装的gRNA表达盒的数量。步骤1:将gRNA寡核苷酸(T#)克隆到单个模块化单gRNA表达载体(pMA-SpCas9-g#)中。步骤2:根据gRNA的总数将单个gRNA表达载体(pMA-T#)组装成1-3个阵列载体。步骤3:对于11-30gRNA表达盒的装配,将2至3个单独的阵列载体进行第二轮组装以产生最终的EGFP表达载体(pMsgRNA-EGFP)。

关键字:CRISPR, SpCas9, Golden-Gate组装, 多重gRNA阵列, 同步遗传操纵

材料和试剂

  1. 200μlPCR管
  2. 1.5 ml Eppendorf管
  3. 10或100μl移液器吸头
  4. 能力 E。大肠杆菌细胞(没有特别的偏好,但应该是重组缺陷型)
  5. 模块gRNA质粒(可从Addgene获得,参见表1中相应的Addgene质粒ID)
  6. 将pFUS-B1至pFUS-B10,pFUS-A,pFUS-A30A和pFUS-A30B质粒(得自Addgene,Golden Gate TALEN和TAL Effector Kit 2.0)(Addgene,目录号:1000000024)
  7. NEB缓冲液2(New England BioLabs,目录号:B7002S)
  8. I(FastDigest)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:FD1014)
  9. T4 DNA连接酶(5U /μl)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:EL0014)
  10. 蒸馏的H 2 O 2 /
  11. 氨苄青霉素(Sigma-Aldrich,目录号:A1593)
  12. LB培养基
  13. dNTP
  14. DreamTaq或其它等同的DNA聚合酶(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:K1072)
  15. 琼脂糖
  16. 质粒制备小试剂盒(不特别优选,我们使用来自MACHEREY-NAGEL的Nucleo Spin质粒easy纯试剂盒,目录号:740727)
  17. 我(BpiI)(FastDigest)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:FD0293)
  18. 质粒安全ATP依赖性DNA酶(Epicentre,目录号:E3101K,ATP包含在该试剂盒中)
  19. 壮观霉素(Sigma-Aldrich,目录号:PHR1441)
  20. X-gal(Sigma-Aldrich,目录号:B4252)
  21. IPTG(溶解于水中的IPTG)(Sigma-Aldrich,目录号:I6758)
  22. I(BspTI)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:FD0834)
  23. I(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:FD0684)
  24. (Thermo Fisher Scientific,Thermo Scientific TM ,目录号:FD0454)的 Bsm I(Esp3I)
  25. 用于PCR筛选的通用引物(参见表1)

    表1.质粒和引物为此方案开发的质粒可从Addgene获得,具有相应的Addgene ID。用于将gRNA寡核苷酸克隆到单个gRNA表达质粒中的单模块质粒(pMA-SpCas9-g1至pMA-SpCas9-10)。用于装配含有11-30个gRNA表达盒的gRNA表达阵列的阵列质粒(pMA-MsgRNA-EGFP)。在该方案中用于筛选组装的阵列质粒的引物序列
    注意:Universal U6和Scr引物已修改,以适应此协议中的PCR条件。

设备

  1. 加热块(Grant Instruments,型号:QBD2)
  2. 微量离心机(Eppendorf,型号:Centrifuge 5424)
  3. 37℃热孵育器(Labnet International,型号:211DS)
  4. 37℃振荡培养箱(环境培养箱摇床G24)(Eppendorf,New Brunswick Scientific,型号:G24)
  5. 热循环仪(Thermo Fisher Scientific,Applied Biosystems ,型号:Veriti 96孔热循环仪)
  6. DNA电泳仪(Bio-Rad Laboratories,型号:Wide mini-sub cell GT)
  7. Nanodrop(Thermo Fisher Scientific,型号:Nanodrop 1000分光光度计)

程序

  1. 准备:设计和订购CRISPR gRNA oligos
    1. 使用任何在线设计工具(如优化的CRISPR设计工具)设计靶gRNA寡核苷酸( http: //crispr.mit.edu/)。
      注意:
      1. 因为gRNA表达盒利用人U6启动子,所以CRISPR靶位点的第一个核苷酸应优选为"G"以确保有效转录。
      2. 排除含有任何以下IIS型限制位点的gRNA寡核苷酸也是非常重要的:BbsI,BsaI和BsmBI。
    2. 对于以"G"开始的靶位点,将以下四个核苷酸突出端添加到有义(SS)和反义(AS)gRNA寡核苷酸:
      SS:5'CACC(N )
      AS:5'AAAC(N )
    3. 对于没有用"G"表示的目标站点,请添加以下悬挂:
      SS:5'CACCG(N )
      AS:5'AAAC(N )C
    4. 从任何合格的公司订购寡核苷酸,稀释寡核苷酸到库存浓度为100 pmol /μl(μM),并存储在-20°C。 (寡核苷酸的标准脱盐纯化是足够的)。

  2. gRNA寡聚物的退火
    1. 退火每个gRNA的有义和反义gRNA寡核苷酸在单独的1.5毫升EP管:
      检测gRNA寡核苷酸:1μl(100pmol /μl)
      反义gRNA寡核苷酸:1μl(100pmol /μl)
      10×NEB缓冲液2:2μl
      加入ddH 2 O至最终体积为20μl。
    2. 在95℃下在加热块中将混合物变性5分钟
    3. 将加热块放在实验台上,缓慢退火寡核苷酸,直到混合物处于室温(约需1-2小时)
    4. 将退火的gRNA寡核苷酸快速离心至管的底部并储存在-20℃以备以后使用或进行以下实验步骤。

  3. 矢量克隆
    使用该方案,在5天(对于1至10gRNA表达盒)至7天(对于11至30盒)完成gRNA表达阵列载体的产生。有关概述,请参见图2中的原理图。在协议中,CRISPR表达阵列中靶位点的位置称为T#(#,表示gRNA的顺序的1至30之间的整数)。该方案利用10个单独的gRNA表达盒,其依赖于盒的数目串联组装成一组"阵列"载体,其中每个阵列载体含有多达10个盒。当组装11-30个盒时,多个阵列向量最终组装成单个向量。用于组装的质粒的选择在步骤C3c,表3中描述。图1中给出了示意图

    图2.协议概述。协议的日常概述。对于组装1至10μgRNA表达盒,在第5天的阳性菌落的小量制备后完成方案。对于装配11至30gRNA表达盒,在第5天进行另一轮组装,并在第7天后完成。 >
    1. 第1天。将gRNA寡核苷酸连接到单个模块化单gRNA表达载体中
      1. 根据下表(针对T1的pMA-SpCas9-g1,针对T11的pMA-SpCas9-g2(针对T11的pMA-SpCas9-g2),针对T11的针对退火的gRNA寡核苷酸T1-TN(N≤30) ,对于T12的pMA-SpCas9-g2,...,对于21的pMA-SpCas9-g1,对于T22的pMA-SpCas9-g2等)(表2)。
        表2.模块化单gRNA表达载体和退火的gRNA寡核苷酸的装配方案该表说明如何选择正确的单一模块质粒(pMA-SpCas9-g1至pMA-SpCas9-10)克隆将gRNA寡核苷酸转化成单个gRNA表达质粒(成功克隆后命名为pMA-T#)。每个gRNA的数目代表gRNA在串联gRNA表达阵列中的位置

      2. 在单独的PCR管中准备装配反应,包含:
        1. 100ng模块化单gRNA表达质粒(pMA-SpCas9-g#)
        2. 1μl来自步骤B4(T#)的退火的gRNA寡核苷酸
        3. 1μlBbsI限制酶
        4. 1μlT4 DNA连接酶
        5. 2μl10×T4连接酶缓冲液(最终浓度为1x)
        6. 无核酸酶水,总反应体积达20μl。
      3. 在以下条件下在热循环仪中孵育反应:


      4. 转化能力。大肠杆菌,使用2μl连接产物。
        注意:用于产生感受态大肠杆菌和转化的方案可在作者请求下获得。
      5. 将板(1/10)转化细胞在含有50μg/ml氨苄青霉素的10cm LB琼脂平板上 注意:不要过度覆盖细胞。
      6. 在37℃下孵育琼脂板过夜。
    2. 第2天。菌落筛选和培养
      1. 使用10或100μl移液器吸头从每次转化中选择两到三个菌落
      2. 将移液器吸头在100μlLB培养基(50μg/ml氨苄青霉素)中开始初始培养。将移液器吸头转移到含有30μl不含核酸酶的水的PCR管中,上下移液管释放细胞。
        注意:初始培养和PCR管应该用相同的ID标记。如果使用超过10 gRNA,我们通常使用两套200μlPCR管,一套用于初始培养,另一套用于细胞裂解液。
      3. 将初始培养管置于37℃培养箱中
      4. 将PCR管置于98℃的热循环仪中10分钟以裂解细胞
      5. 进行细胞裂解物的gRNA PCR筛选。添加到每个反应:
        1. 1μl转化的大肠杆菌裂解物
        2. 5 pmol通用U6正向引物(表1)
        3. 5pmol所讨论的gRNA的反义寡核苷酸
        4. 0.4μldNTP(10mM)
        5. 0.1μlDreamTaq聚合酶(或类似物)
        6. 2μl10x DreamTaq缓冲液(最终浓度为1x)
        7. 无核酸酶水至20μl总反应体积。
        实施例:对于pMA-SpCas9-T1的菌落筛选,使用通用U6启动子作为正向引物和T1反义寡核苷酸作为反向引物对2-3个挑选的菌落进行PCR。
      6. 在热循环仪中用以下条件孵育PCR反应物:


      7. 通过电泳在1%琼脂糖凝胶上显现PCR产物。预期的扩增子大小约为270bp
      8. 从每个转化中选择一个阳性菌落,并将50μl初始培养物转移到含有5ml含有50μg/ml氨苄青霉素的LB培养基的单个管中。
      9. 在37℃在轨道摇床中孵育过夜。
    3. 第3天。质粒制备和单个gRNA表达盒装配到'阵列'质粒
      1. 使用商业小量制备试剂盒根据制造商的说明书从过夜培养物中分离质粒DNA(命名为pMA-T#)。
      2. 使用通用U6引物(推荐)通过Sanger测序验证单个gRNA表达质粒。
      3. 根据表3选择用于单个gRNA表达质粒的1种组装的阵列质粒:

        表3.选择用于装配pMA-T#载体的阵列质粒。阵列质粒的选择取决于待装配的gRNA表达盒的数量。对于1-10μgRNA盒的装配(左),根据gRNA盒的数目选择pFUS-B#阵列质粒之一。当装配11-20μgRNA盒(中间)时,选择pFUS-A用于装配gRNA盒1至10.对于剩余的盒(≤10),根据数目选择一个pFUS-B#阵列质粒gRNA盒。对于21-30gRNA盒的装配(右),pFUS-A30A用于装配前10个gRNA表达盒。 pFUS-A30B用于装配gRNA盒11至20.根据gRNA盒的数目将剩余的盒(≤10)克隆到pFUS-B#阵列质粒中。


      4. 在单独的PCR管中设置金门装配反应,包含:
        1. 30 ng阵列质粒 注意:质粒数量已优化。
        2. 50ng各个单独的模块化单gRNA质粒(最多10个质粒)
        3. 1μl Bsa I限制酶
        4. 1μlT4 DNA连接酶
        5. 2μl10×T4连接酶缓冲液(最终浓度为1x)
        6. 无核酸酶水至20μl总反应体积。
      5. 在以下条件下在热循环仪中孵育反应:


      6. 加入1μl25 mM ATP和1μl质粒安全DNase进行反应 注意:质粒安全DNase处理消化所有未连接的线性dsDNA片段,不完整的装配产物和线性化载体。该步骤使感受态细胞中线性化dsDNA片段的体内重组的风险最小化。当组装五个以上的gRNA表达盒时,这一步骤是非常重要的。
      7. 在37℃下,在热循环仪中孵育反应1小时
      8. 转化能力。大肠杆菌细胞,使用2μl组装产物
      9. 在含有50μg/ml壮观霉素以及8μl100mg /μlX-gal和8μl0.5M IPTG的10cm LB琼脂平板上转化细胞(1/20),用于蓝色/白色菌落筛选。 >
      10. 在37℃下孵育琼脂板过夜。
    4. 第4天。


      菌落筛选和培养
      1. 检查板的阳性白色菌落。白色与蓝色菌落的预期比率对于10μgRNA表达盒的成功装配至少为25%,并且随着gRNA表达盒数量的减少而逐渐增加(图3)。


        图3.第一轮装配的检查和筛选左图:M10的变形板。白色菌落含有插入的载体。蓝色菌落包含空载体。右:8个白色菌落的M10筛选的通用PCR。泳道1-4和7-8显示了类似于指示阳性菌落的梯度的多个条带。泳道5和6仅显示没有梯形图的两个主导谱带,表示不正确的插入
      2. 从第2天所述的每次转化中选择两到三个白色菌落。
      3. 设置PCR筛选反应的所有挑选的菌落在单独的PCR管,包含:
        1. 1μl转化的E。大肠杆菌裂解物
        2. 5 pmol通用U6正向引物(表1)
        3. 5 pmol通用Scr反向引物(表1)
        4. 0.4μldNTP
        5. 0.1μlDreamTaq聚合酶(或类似物)
        6. 2μl10x DreamTaq缓冲液(最终浓度为1x)。
        7. 无核酸酶水至20μl总反应体积
      4. 在热循环仪中用以下条件孵育PCR反应物:


      5. 通过电泳在1%琼脂糖凝胶上显现PCR产物。正确的扩增模式应该是具有在约400bp的显性条带的"梯形",并且对应于包含在扩增中的每个额外的gRNA表达盒的每个"步骤"增加392bp(图3B)。
      6. 从每次转化中选择一个阳性菌落,将50μl初始培养物转移到含有5ml含50μg/ml壮观霉素的LB培养基的单独管中。
      7. 在37℃在轨道摇床中孵育过夜。
    5. 第5天。用于含有11至30个表达盒的质粒的质粒DNA制备和2
      1. 使用商业小量制备试剂盒根据制造商的说明书从过夜培养物中分离质粒DNA。
      2. 通过限制酶消化验证装配的阵列质粒。在1.5 ml EP管中,加入:
        1. 0.5μg装配的阵列质粒DNA
        2. 1μl Af II限制酶
        3. 1μlXba I限制酶
        4. 2μl10x反应缓冲液(最终浓度为1x)
        5. 无核酸酶水至20μl总反应体积。
      3. 通过电泳在1%琼脂糖凝胶上显示消化产物 注释:对于10个盒(〜400bp/gRNA表达盒),释放的表达盒的预期大小为约4kb。我们强烈建议为每个多重载体生成理论序列图。如果在任何gRNA指南序列中存在AflII或XbaI识别位点,则RFLP结果将不同。
      4. 对于组装的阵列质粒的进一步验证,为个别PCR管中阵列载体中存在的所有gRNA表达盒设置引导特异性PCR。
        添加到每个PCR反应:
        1. 1ng阵列质粒DNA
        2. 5 pmol pMA-TN中特异性gRNA的正义寡核苷酸作为正向引物
        3. 5pmol pMA-TN + 1中下一特异性gRNA的反义寡核苷酸作为反向引物
        4. 0.4μldNTP
        5. 0.1μlDreamTaq聚合酶(或类似物)
        6. 2μl10x DreamTaq缓冲液(最终浓度为1x)。
        7. 无核酸酶水至20μl总反应体积。
        实施例:为了验证组装的pFUS-B4-M4,应进行三个单独的PCR反应。第一个是将T1的SS寡核苷酸作为正向引物,将T2的AS寡核苷酸作为反向引物。第二个是T2的SS寡核苷酸作为正向引物,T3的AS寡核苷酸作为反向引物。第三个是T3的SS寡核苷酸作为正向引物,T4的AS寡核苷酸作为反向引物。每个装配阵列质粒的PCR反应总数为N-1(N是阵列质粒中包含的gRNA表达盒的数目)(图4)。
      5. 在热循环仪中用以下条件孵育PCR反应物:


      6. 通过电泳在1%琼脂糖凝胶上显现PCR产物。预期的扩增子大小约为400bp

        图4.指导特异性PCR验证。上图:用于指导特异性PCR的引物的示意图。下图:使用在1%琼脂糖凝胶上可视化的pFUS-B4-M4质粒的指导特异性PCR的实例。

      7. 在PCR管中设置阵列质粒的2 nd 组装(仅用于装配11-30个盒),其包含:
        1. 每个装配的阵列载体(pFUS-A-M10 + pFUS-B#-M#或pFUS-A30A-M10 + pFUS-A30B-M10 + pFUS-B#-M#)的50ng DNA。
        2. 50ng阵列质粒pMsgRNA-EGFP的DNA(表1)
        3. 1微升Bsm B I限制酶
        4. 1μlT4 DNA连接酶
        5. 2μl10×T4连接酶缓冲液(最终浓度为1x)
        6. 无核酸酶水至20μl总反应体积
      8. 在热循环仪中用以下条件孵育反应物:


      9. 转化能力。大肠杆菌细胞用2μl连接产物
      10. 在含有50μg/ml氨苄青霉素以及40μl20mg/ml X-gal和40μl0.1M IPTG(用于蓝/白菌落筛选)的10cm LB琼脂平板上平板(1/10)转化的细胞。
      11. 孵育琼脂平板在37℃过夜。
    6. 第6天。3 菌落筛选和培养
      1. 检查板为阳性白色菌落。白色与蓝色菌落的预期比率为> 80%,以便成功组装
      2. 按第2天所述选择2至3个白色菌落。
      3. 为所有挑选的菌落的gRNA表达盒的每个组装部分设置重叠PCR筛选反应。对于个别PCR管,添加:
        1. 1μl转化的E。大肠杆菌裂解物
        2. 5pmol作为正向引物的第一或第二表达盒阵列中最后一个gRNA(T10或T20)的正义寡核苷酸。
        3. 5 pmol第二或第三表达盒阵列中第一gRNA(T11或T21)的反义寡核苷酸作为反向引物。
        4. 0.4μldNTP
        5. 0.1μlDreamTaq聚合酶(或类似物)
        6. 2μl10x DreamTaq缓冲液(最终浓度为1x)。
        7. 无核酸酶水至20μl总反应体积。
        实施例:为了验证组装的pMsgRNA-EGFP-M30(含有从pFUS-A30A-pFUS-A30B-pFUS-B10的装配产生的30μgRNA表达盒的阵列质粒),进行两个单独的重叠PCR反应。第一反应使用T10的有义(SS)寡核苷酸作为正向引物,使用T11的反义(AS)寡核苷酸作为反向引物。第二种使用T20的SS寡核苷酸作为正向引物和T21的AS寡核苷酸(图5)。
      4. 在热循环仪中用以下条件孵育PCR反应物:


      5. 通过电泳在1%琼脂糖凝胶上显现PCR产物。预期的扩增子大小约为460bp
      6. (可选)对所有邻近的gRNA执行重叠PCR

        图5.重叠PCR筛选:上图:pMsgRNA-EGFP-M30的重叠PCR筛选的示意图。底部:在1%琼脂糖凝胶上显现的pMsgRNA-EGFP-M30的指导特异性PCR验证的实例。泳道10和泳道20含有上述重叠PCR反应。注意,重叠PCR产生比引物特异性PCR稍大的条带。强烈建议生成理论质粒序列图作为装配,PCR验证和限制性酶消化的指南
      7. (可选)使用T#作为引物的Sanger测序。
      8. 选择一个阳性菌落并将50μl初始培养物转移到含有5ml添加有50μg/ml氨苄青霉素的LB培养基的各管中。
      9. 在37℃在轨道摇床中孵育过夜
    7. 第7天。质粒DNA的制备和验证
      1. 根据制造商的说明书,使用商业小量制备试剂盒从过夜培养物中分离质粒DNA
      2. 通过限制酶消化验证组装的pMsgRNA-EGFP-M#阵列质粒,以释放组合的gRNA表达阵列盒。加入PCR管中:
        1. 0.5μg装配的阵列质粒DNA
        2. 1μlSfi I限制酶。
        3. 2μl10x反应缓冲液(最终浓度为1x)
        4. 无核酸酶水至20μl总反应体积
      3. 通过电泳在1%琼脂糖凝胶上显示消化产物。释放的表达盒的预期大小为约1662 + N×379bp(N为最终阵列质粒中含有的gRNA表达盒的总数)。

笔记

  1. 为了在每个装配反应中克隆的效率,对于单个gRNA表达载体,我们通常经历高于95%的阳性率。该阳性率基于PCR筛选。 gRNA表达阵列的装配的阳性率基于白色(阳性)与蓝色(阴性)菌落的比率。请注意,所有蓝色菌落都是阴性。然而,如果白色菌落含有不正确的连接或重组产物,则它们也可以是阴性的。因此,进行PCR筛选是重要的。
  2. 如果观察到高的蓝色菌落率,建议检查酶的有效期,因为FastDigest酶具有相对短的储存时间。其他措施如降低装配反应中pFUS质粒的量,增加循环数,增加酶的量可以提高白色菌落率。

致谢

JVN和ALN得到Lundbeck基金会,Krista Og Viggo Petersens Fond,Fabrikant Einar Willumsens Mindelegat和FondenLægevidenskabensFremme的资助。 LL和LB由Lundbeck基金会的DREAM项目提供支持。 YL由丹麦独立研究研究委员会,Sapere Aude Young研究人才奖YL,Lundbeck基金会和丹麦创新基金(BrainStem)资助。该方案是基于我们先前发表在"细胞分子生命科学"(Vad-Nielsen等人,2016)中的研究而开发的。我们还认可Voytas DF的研究小组,他们为本方案中使用的第一个装配构建了质粒(Cermak等人,2011)。

参考文献

  1. Cermak,T.,Doyle,EL,Christian,M.,Wang,L.,Zhang,Y.,Schmidt,C.,Baller,JA,Somia,NV,Bogdanove,AJand Voytas,DF(2011) 高效设计和装配定制TALEN和其他基于TAL效应的构建体 用于DNA靶向。核酸研究 39(12):e82。
  2. Vad-Nielsen,J.,Lin,L.,Bolund,L.,Nielsen,AL和Luo,Y。(2016)。  用于同时靶向多个基因的CRISPR gRNA表达阵列的金门装配 Cell Mol Life Sci 73 :4315-4325。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Vad-Nielsen, J., Lin, L., Jensen, K. T., Nielsen, A. L. and Luo, Y. (2016). A Golden Gate-based Protocol for Assembly of Multiplexed gRNA Expression Arrays for CRISPR/Cas9. Bio-protocol 6(23): e2059. DOI: 10.21769/BioProtoc.2059.
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