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Heterologous Expression and Purification of the CRISPR-Cas12a/Cpf1 Protein
CRISPR-Cas12a/Cpf1的异源表达和纯化   

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Molecular Cell
Apr 2017

Abstract

This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli. It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for purification of other Cas12a homologs and the purified proteins can be used for subsequent genome editing experiments.


Figure 1. Timeline of activities for the heterologous expression and purification of Francisella novicida Cas12a (FnCas12a) from Escherichia coli

Keywords: CRISPR-Cas (CRISPR-Cas), Cas12a (Cas12a), Cpf1 (Cpf1), Protein purification (蛋白质纯化)

Background

Prokaryotic CRISPR-Cas immune systems provide protection against viruses and plasmids by using CRISPR RNAs (crRNAs) as a guide for sequence-specific targeting of foreign DNA or RNA (van der Oost et al., 2014; Marraffini, 2015). Class 1 CRISPR-Cas systems (comprising types I, III, and IV) typically form multi-subunit protein-crRNA effector complexes, while the class 2 systems (comprising types II, V, and VI) rely on single crRNA-guided effector nucleases for target interference (Mohanraju et al., 2016).

Effector nuclease enzymes from the Class 2 CRISPR-Cas systems have emerged as efficient and precise tools for genome editing and gene expression control (Mali et al., 2013; Doudna and Charpentier, 2014; Hsu et al., 2014). The widely used Cas9, which is the signature protein of type II systems, utilizes a dual guide RNA structure consisting of crRNA and a trans-activating crRNA (tracrRNA) for target recognition (Deltcheva et al., 2011). For genome editing purposes, the dual guide RNA is often replaced by a synthetic fusion of the mature crRNA and tracrRNA, resulting in a long single-molecule guide RNA (sgRNA) in which the individual RNAs are fused by a short linker sequence (Jinek et al., 2012). The sequence of the guide RNA allows binding of complementary DNA targets by base pairing with the target strand, while the other strand of the DNA is displaced. Upon finding a cognate DNA target, the HNH and RuvC nuclease domains of Cas9 mediate cleavage of the target and the displaced strand, respectively (Jinek et al., 2012; Karvelis et al., 2013).

More recently, another novel class 2 CRISPR-Cas nuclease with distinctive features has been identified in bacterial genomes: Cas12a (also known as Cpf1) (Makarova and Koonin, 2015; Zetsche et al., 2015; Shmakov et al., 2017). Cas12a utilizes a single crRNA guide for DNA targeting; it does not require a tracrRNA, resulting in a shorter gRNA sequence compared to the chimeric single-molecule guide RNAs (sgRNA) used by Cas9. While Cas9 requires RNase III-mediated processing of pre-crRNA or individual expression of sgRNAs for the formation of mature guide RNAs, Cas12a can process its own pre-crRNA. This pre-crRNA processing activity allows for simple multiplexing in Cas12a-mediated genome editing (Wang et al., 2017; Zetsche et al., 2017). Whereas Cas9 generates double stranded DNA breaks (DSBs) that are blunt ended, Cas12a generates staggered-end DSBs (Zetsche et al., 2015). Such overhangs can be utilized for overhang-based cloning (Li et al., 2016; Lei et al., 2017). Moreover, Cas9 typically recognizes a G-rich PAM sequence, while all Cas12a orthologues characterized to date recognize a T-rich PAM sequence (Zetsche et al., 2015). Taken together, these features make Cas12a a valuable addition to the genome editing toolbox.

Cas12a has been successfully repurposed for genome editing applications in mammalian cells (Zetsche et al., 2015; Kim et al., 2016a), mice (Hur et al., 2016; Kim et al., 2016b), rice (Endo et al., 2016; Hu et al., 2017; Xu et al., 2017), yeast (Verwaal et al., 2017; Swiat et al., 2017), zebrafish, xenopus (Moreno-Mateos et al., 2017), microalga (Ferenczi et al., 2017) and plant cells (Zaidi et al., 2017; Kim et al., 2017; Tang et al., 2017). The high efficiency and specificity of Cas12a in human cells, coupled with fewer off-target cleavage events compared to Cas9 (Kleinstiver et al., 2016), makes Cas12a a robust and reliable tool for genome editing.

For its in vitro characterization and crystallization (Swarts et al., 2017), Cas12a from Francisella novicida U112 was purified after heterologous expression in Escherichia coli. The expression strain E. coli RosettaTM 2 (DE3) carries a chromosomal T7 RNA polymerase gene under control of an IPTG inducible lacUV5 promoter. The cas12a gene is expressed using a pET vector (Studier and Moffatt, 1986; Rosenberg et al., 1987; Studier et al., 1990) with a lacI-controlled T7 promoter. Here we describe the steps required for controlled expression and purification of FnCas12a. The protocol can also be used for the expression and purification of Cas12a homologs from Acidaminococcus sp. and Lachnospiraceae bacterium.

Materials and Reagents

Note: Equivalent materials and reagents may be used as substitutes.

  1. Expression of FnCas12a in E. coli RosettaTM 2(DE3)
    1. 100-ml Erlenmeyer flask (DWK Life Sciences, DURAN®, catalog number: 21 213 24 )
    2. 2-L Erlenmeyer flask (DWK Life Sciences, DURAN®, catalog number: 21 216 63 )
    3. 5-L Erlenmeyer flasks (DWK Life Sciences, DURAN®, catalog number: 21 216 73 )
    4. 50-ml conical centrifuge tubes (Sigma-Aldrich, catalog number: T2318-500EA )
    5. 2-ml screw top tube (Corning, catalog number: 430659 )
    6. NalgeneTM PPCO Centrifuge Bottles with Sealing Closure (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3141-0500 ) or equivalent 500-ml centrifuge bottles
    7. Pipette tips (DeckWorksTM standard pipet tips, Corning, catalog numbers: 4110 ; 4112 ; 4867 )
    8. 10-ml syringe (BD, catalog number: 309604 )
    9. 0.22 μm syringe filter (Mdi, catalog number: SYPL0601MNXX204 )
    10. 250-ml bottle (Greiner Bio One International, catalog number: 227261 )
    11. Escherichia coli RosettaTM 2(DE3) cells (Merck, Novagen, catalog number: 71400 ) [encodes a T7 RNA polymerase gene under control of a lacUV5 promoter]
    12. Plasmid pDS015* [pET His6 TEV LIC cloning vector (Addgene, catalog number: 29653 ), with F. novicida U112 cas12a gene insert fused to an N-terminal His-tag; expression under the control of a lacI-controlled T7 promoter]
      *Note: Acidaminococcus sp. BV3L6 Cas12a (AsCas12a) and Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) proteins can also be purified using this protocol with expression vectors 6His-MBP-TEV-huAsCpf1 (Addgene, catalog number: 90095 ) and 6His-MBP-TEV-huLbCpf1 (Addgene, catalog number: 90096 )
    13. Tryptone (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: LP0042B )
    14. Yeast extract (BD, BactoTM, catalog number: 212720 )
    15. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-10 )
    16. Sodium hydroxide (NaOH) (Merck, EMD Millipore, catalog number: 106462 )
    17. Ethanol (Fisher Scientific, catalog number: BP2818500 )
    18. Chloramphenicol (Fisher Scientific, catalog number: BP904100 )
    19. Kanamycin sulfate (Thermo Fisher Scientific, catalog number: 11815024 )
    20. Glycerol (Fisher Scientific, catalog number: BP229-4 )
    21. IPTG (Fisher Scientific, catalog number: BP1755-1 )
    22. Agar (Acros Organics, catalog number: 400400050 )
    23. LB medium (see Recipes)
    24. 1,000x chloramphenicol solution (34 mg/ml) (see Recipes)
    25. 1,000x kanamycin solution (50 mg/ml) (see Recipes)
    26. 1 M IPTG (IsoPropyl-1-Thio-β-D-Galactopyranoside) (see Recipes)
    27. Glycerol stock (50% solution) (see Recipes) 

  2. Purification of FnCas12a
    1. 5 ml HisTrap HP (GE Healthcare, catalog number: 17524701 )
    2. Dialysis tubing, high retention seamless cellulose tubing, avg. flat width 23 mm (0.9 in.), MWCO 12,400, 99.99% retention (Sigma-Aldrich, catalog number: D0405 )
    3. Dialysis tubing clamps (Sigma-Aldrich, catalog number: Z371092 )
    4. 5 ml HiTrap Heparin HP (GE Healthcare, catalog number: 17040601 )
    5. Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-100 membrane (Merck, EMD Millipore, catalog number: UFC9100 )
    6. HiLoad 16/600 Superdex 200 pg (GE Healthcare, catalog number: 28989335 )
    7. GosselinTM Round-Base 10-ml Test Tubes (Corning, GosselinTM, catalog number: TP10-01 ) or other equivalent fraction collection tubes
    8. NalgeneTM Oak Ridge High-Speed Centrifuge Tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3114-0050 ) or equivalent 50-ml centrifuge tubes
    9. Membrane Filter, mixed cellulose esters (Merck, MF-Millipore, catalog number: HAWP04700 )
    10. Membrane Filter, mixed cellulose esters (Merck, MF-Millipore, catalog number: GSWP04700 )
    11. Cell pellet from overnight culture in which FnCas12a was expressed (from Procedure A)
    12. cOmpleteTM, EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, Roche Diagnostics, catalog number: 11873580001 )
    13. Lysozyme from chicken egg white (Sigma-Aldrich, catalog number: L6876-5G )
    14. β-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
    15. TEV protease (Sigma-Aldrich, catalog number: T4455 )
    16. 12% Mini-PROTEAN® TGXTM Precast Protein Gels (Bio-Rad Laboratories, catalog number: 4561043 )
    17. 4x Laemmli protein sample buffer for SDS-PAGE (Bio-Rad Laboratories, catalog number: 1610747 )
    18. Bio-SafeTM Coomassie Stain (Bio-Rad Laboratories, catalog number: 1610786 )
    19. PageRuler Prestained Protein Ladder, 10 to 250 kDa (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 26619 )
    20. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
    21. Ethylenedinitrilotetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
    22. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-10 )
    23. Tris (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 17926 )
    24. Imidazole (Sigma-Aldrich, catalog number: I0250 )
    25. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
    26. Potassium chloride (KCl) (Merck, EMD Millipore, catalog number: 104933 )
    27. HEPES (Sigma-Aldrich, catalog number: H3375 )
    28. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 757551 )
    29. Glycine (Sigma-Aldrich, catalog number: G8898 )
    30. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
    31. 1 M DTT (Dithiothreitol) stock (see Recipes)
    32. 0.5 M EDTA (Disodium Ethylene Diamine Tetra-Acetate) stock (pH 8) (see Recipes)
    33. Lysis Buffer (see Recipes)
    34. Wash Buffer (see Recipes)
    35. Elution Buffer (see Recipes)
    36. Dialysis Buffer (see Recipes)
    37. Dilution Buffer (see Recipes)
    38. IEX-A Buffer (see Recipes)
    39. IEX-B Buffer (see Recipes)
    40. SEC Buffer (see Recipes)
    41. 10x SDS-PAGE Electrophoresis Running Buffer (see Recipes)

  3. Activity assay using purified Cas12a
    1. Purified Cas12a Nuclease (from Procedure B)
    2. Nuclease-free water
    3. Proteinase K, Molecular Biology Grade (New England Biolabs, catalog number: P8107S )
    4. crRNA containing the targeting sequence complementary to the target DNA
      Note: The RNA can be ordered as a desalted RNA oligonucleotide or as PAGE-purified RNA oligonucleotide from an RNA synthesis company such as Sigma-Aldrich or IDT.
    5. DNA substrate containing the target sequence and a 5’ TTTN PAM sequence
      Note: The substrate DNA can be circular or linearized plasmid, PCR products, or synthesized oligonucleotides). As an example, the DNA substrate and crRNA used in the activity assay is shown in Figure 2.


      Figure 2. Schematic of the Cas12a crRNA-DNA-targeting complex. The expected cleavage sites are indicated by red arrows.

    6. GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SM0311 ) or equivalent
    7. DNA gel Loading Dye [e.g., 6x DNA Loading Dye (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0611 )]
    8. InvitrogenTM SYBRTM Safe DNA Gel Stain (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: S33102 )
    9. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-10 )
    10. Magnesium chloride hexahydrate (MgCl2·6H2O)
    11. HEPES (Sigma-Aldrich, catalog number: H3375 )
    12. Ethylenedinitrilotetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
    13. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
    14. 10x Nuclease Reaction Buffer (see Recipes)

Equipment

Note: Equivalent equipment can be used.

  1. Expression of FnCas12a in E. coli RosettaTM 2
    1. Pipettes (Corning, model: LambdaTM Plus Single-Channel Pipettor, catalog numbers: 4070 ; 4074 ; 4075 )
    2. New BrunswickTM Innova® 42 incubator (Eppendorf, New BrunswickTM, model: Innova® 42 , catalog number: M1335-0002) or an equivalent incubator that can be set at 37 °C
    3. Sorvall LYNX 4000 Superspeed Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall LYNX 4000 , catalog number: 75006580) or an equivalent centrifuge that can be cooled down to 4 °C and can perform up to 6,000 x g
    4. New BrunswickTM Innova® 44/44R (Eppendorf, New BrunswickTM, model: Innova® 44/44R , catalog number: M1282-0002) or any equivalent shaker incubator where the temperature can be set at 37 °C and 18 °C
    5. Cell density meter (GE Healthcare, model: UltrospecTM 10 , catalog number: 80-2116-30), or equivalent spectrophotometer that can measure the density of cells in suspension at 600 nm
    6. Ice-water bath (water and ice mixed)

  2. Purification of FnCas12a
    1. SONOPULS HD (Bandelin electronic, model: HD 3200 ) with VS 70 T Sonotrode (Bandelin) or equivalent ultrasonic homogenizer/Sonifier, or alternatively a French Pressure Cell (French Press) for cell lysis
    2. Peristaltic pump P-1 with connectors for 5 ml HisTrap HP (GE Healthcare, model: Peristaltic Pump P-1, catalog number: 18111091 ) or an equivalent peristaltic pump
    3. Tubing Connectors for Use with Peristaltic Pump P-1 (GE Healthcare, catalog number: 11300082 )
    4. ÄKTApurifier 10 FPLC system (GE Healthcare, model: ÄKTApurifier 10 , catalog number: 28406264) or an equivalent FPLC system
    5. Sorvall LYNX 4000 Superspeed Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall LYNX 4000 , catalog number: 75006580) or an equivalent centrifuge that can be cooled down to 4 °C and can perform up to 30,000 x g
    6. pH meter (QiS, model: B210 )
    7. Filter holder assembly for filtration (Merck, catalog number: XX1014700 or Nalgene, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: DS0320-2545 ), or equivalent filter holder assembly
    8. Diaphragm Vacuum Pumps LABOPORT® N 820 (ABM van Zijl B.V, catalog number: ABMK N8203FT18 ), or an equivalent vacuum pump
    9. Nanodrop (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 , catalog number: ND-2000)
    10. Mini-PROTEAN Tetra cell (Bio-Rad Laboratories, model: Mini-PROTEAN Tetra Cell, catalog number: 1658004EDU ), or an equivalent vertical electrophoresis system
    11. Epson Perfection V850 Pro scanner (Epson, model: Perfection V850 Pro ) or equivalent scanner or imager suitable for SDS-PAGE gel imaging.

  3. Activity assay using purified Cas12a
    1. EppendorfTM 5424 Microcentrifuge (Eppendorf, model: 5424 , catalog number: 022620498)
    2. MUPID One Horizontal Electrophoresis System (Bulldog Bio, catalog number: MU2 ) or an equivalent horizontal electrophoresis system
    3. G:BOX F3 (Syngene, model: G:BOX F3 , catalog number: 05-GBOX-F3) gel doc system or equivalent DNA agarose gel imaging equipment

Procedure

  1. Transformation of E. coli RosettaTM 2(DE3) with pDS015 plasmid and preparation of a glycerol stock
    1. Add 1 ng of pDS015 plasmid DNA directly to 50 µl of chemically competent E. coli RosettaTM 2(DE3) cells.
    2. Stir gently to mix and place the tubes on ice for 5 min.
    3. Heat the tubes for exactly 30 sec in a 42 °C water bath; do not shake.
    4. Immediately place the tube on ice for 2 min.
    5. Add 250 μl of room temperature sterile SOC medium (provided by the manufacturer) to the tube. Incubate at 37 °C while shaking at 250 rpm for 60 min.
    6. Spread 5-50 μl of the transformation mixture on LB agar plates containing 34 µg/ml and 50 µg/ml of chloramphenicol and kanamycin, respectively. If plating less than 25 μl of the transformation, we recommend adding 50 µl of sterile SOC medium to the transformation mixture before plating to facilitate even colony distribution on the LB agar plate surface.
    7. Incubate the LB agar plates overnight at 37 °C.
    8. The next day, pick a single colony from the transformation plates using a sterile pipette tip and inoculate 10 ml LB in a 50-ml tube.
    9. Incubate the 50-ml tube overnight in a 37 °C shaking incubator shaking at 160 rpm.
    10. The next day, add 500 μl of the overnight culture to 500 μl of 50% sterile glycerol in a 2-ml screw-top tube or cryovial and mix gently.
    11. Store the glycerol stock at -80 °C for future use.

  2. Large-scale expression of FnCas12a in E. coli RosettaTM 2(DE3)
    Day 1. Preparation of media, buffers and single colonies
    1. Prepare 20 ml of LB in a 100-ml Erlenmeyer flask (for starting overnight cultures) (Recipe A1).
    2. Prepare three 5-L Erlenmeyer flasks, each containing 1.5 L of LB medium (for large-scale propagation and protein purification) (Recipe A1).
    3. Prepare antibiotic and stock solutions (Recipes A2 and A3).
    4. Prepare the buffers needed for the purification (Recipes B1-B8).
    5. Streak out a glycerol stock of E. coli RosettaTM 2(DE3) transformed with pDS015 on an LB agar plate containing 50 μg/ml kanamycin and 34 μg/ml chloramphenicol.
    6. Incubate the LB agar plate overnight in a 37 °C incubator.

    Day 2. Overnight culture preparation
    1. Add 20 μl of the 50 mg/ml kanamycin stock solution and 20 μl of the 34 mg/ml chloramphenicol stock solution to the 20 ml of autoclaved LB medium in a 100-ml Erlenmeyer flask from Day 1.
    2. With a sterile pipette tip, pick a single colony of E. coli RosettaTM 2(DE3) transformed with pDS015 from the LB agar plate from Day 1.
    3. Use the colony to inoculate the medium containing the kanamycin and chloramphenicol.
    4. Loosely close the 100-ml Erlenmeyer flask with a cotton plug.
    5. Incubate the bacterial culture at 37 °C for 16-20 h in a shaking incubator (set at 160 rpm).

    Day 3. Large-scale propagation of cells overexpressing FnCas12a
    1. Take three autoclaved 5-L Erlenmeyer flasks* each containing 1.5 L LB medium from Day 1.
    2. To each flask, add 1.5 ml of 50 mg/ml chloramphenicol solution.
    3. To each flask, add 1.5 ml of 34 mg/ml kanamycin solution.
    4. To each flask, add 15 ml of the overnight culture prepared on Day 2.
    5. Incubate the culture flasks at 37 °C in a shaking incubator at 160 rpm**.
    6. Monitor the OD600 nm of the culture every half an hour. Once an OD600 nm of 0.5-0.6 is reached (this normally takes ~3-4 h), transfer the Erlenmeyer containing the culture to the ice-water bath and incubate (cold-shock) it for 15 min. This step slows down the metabolism of E. coli and triggers expression of cold-shock proteins which may aid FnCas12a folding during expression.
    7. To each flask, add 200 μl of filter-sterilized 1 M IPTG solution to the culture to induce expression of FnCas12a.
    8. Transfer the culture to an 18 °C shaking incubator (set at 120 rpm) for overnight expression (~16 h).
      Notes:
      1. *It is also possible to express smaller volumes of cell culture (e.g., a single 1.5 L culture or one or more 750 ml cultures in 2-L Erlenmeyer flasks)
      2. **When using a baffled Erlenmeyer flask, reduce the shaking incubator speed to 120 rpm to prevent the formation of foam. 

    Day 4–Part I. Large-scale propagation of cells overexpressing FnCas12a (continued)
    1. Transfer the overnight culture from Day 3 to centrifuge bottles.
    2. Harvest the cells by centrifuging the culture for 15 min at 6,000 x g at 4 °C.
    3. Discard supernatant and store the pelleted cells at -20 °C (for use within a week for optimal purification) or at -80 °C (for long-term storage) or proceed directly to purification. The expected yield is ~5 g of cell pellet per liter of cell culture.

    Day 4–Part II. Purification of FnCas12a–Part I
    1. If continuing with a frozen cell pellet, thaw the cell pellet from Day 4–Part I on ice for 30-60 min. If proceeding directly after protein expression, skip this step.
      Note: All subsequent steps should be performed on ice or at 4 °C.
    2. Resuspend the entire cell pellet in Lysis Buffer (~2.5-5 ml Lysis Buffer per gram of cell pellet).
    3. Add 1 tablet cOmpleteTM protease inhibitor for every 50 ml.
    4. Add lysozyme to a final concentration of 1 mg/ml.
    5. Incubate the sample on ice for 30 min.
    6. If using a French Press for cell lysis: after the lysozyme treatment, pass cell suspension through French Press twice at 16,000 psi.
    7. If using sonication for cell lysis: after the lysozyme treatment, lyse the cell suspension by using a sonicator with an appropriate tip and a protocol suitable for lysis of large volume cell suspensions. For our setup (Bandelin SONOPULS HD with VS 70 T tip), we use the following settings: 10 min total time, 1 sec on, 0.7 sec off, and 20% amplitude. The cell suspension often has a brownish tinge after lysis as shown in Figure 3.
      Note: Using a French Pressure cell or sonicator gives approximately the same yield–FnCas12a is very stable and little to no protein will be lost during sonication. Keep in mind, however, that sonication is usually less suitable for large volumes, and therefore a protocol suitable for lysis of a large volume of cell suspension should be applied.


      Figure 3. After lysis, the cell suspension becomes tinted brown and less viscous

    8. Pour the lysate into (a) centrifugation tube(s) and centrifuge for 45 min, 4 °C, at 30,000 x g.
    9. Transfer the supernatant to (a) clean 50-ml tube(s). This is the ‘cell-free extract’.
      Note: A sample of the lysed cell pellet may be stored at 4 °C and analyzed later by SDS-PAGE analysis for the presence of FnCas12a to assess its solubility and to determine if cell lysis was successful.
    10. Pass the cell free extract through a 0.22 µm membrane filter and save the filtrate into a sterile tube.
    11. Using a peristaltic pump, wash a HisTrap HP column with 3-5 column volumes of distilled water to remove the solution in which the resin is stored.
    12. Using a peristaltic pump, equilibrate the HisTrap HP with at least 5 column volumes of Lysis Buffer with a flow rate of 2 ml/min.
    13. Using the peristaltic pump*, pass the filtered cell free extract with a flow rate of 1 ml/min through the HisTrap HP column and collect the flow-through in (a) 50-ml tube(s) labeled ‘flow-through’**.
      Notes:
      1. *For sample loading, a superloop can be used instead of the peristaltic pump.
      2. **A sample of the flow-through may be stored at 4 °C and analyzed later by SDS-PAGE analysis for the presence of the FnCas12a to determine if the protein bound to the column (if a large fraction of the protein remains in the flow-through, regenerate or replace your column). In some cases, the high amount of proteins can saturate the column. To recover the protein in the flow-through, the flow-through can be (re)loaded onto another or regenerated HisTrap HP column. 

    The next chromatography steps are performed using an ÄKTA FPLC system.
    1. Equilibrate the ÄKTA FPLC system with Wash Buffer until the absorbance at 280 nm reaches a steady baseline. Transfer the HisTrap HP column to the ÄKTA FPLC and wash the column using Wash Buffer with a flow rate of 2 ml/min for 10-15 times the column volume or until the absorbance at 280 nm becomes near** stable with the Wash Buffer. Collect the first 3-4 10-ml wash fractions* in separate tubes labeled ‘wash-through #’.
      Notes:
      1. *The wash-through may be saved and checked later using SDS-PAGE analysis for the presence of FnCas12a to determine if it was eluted off the column during washing.
      2. **Even at its low concentration, imidazole in the washing buffer can remove small amounts of the protein of interest. Therefore, start eluting the protein as the absorbance at 280 nm is near stable to avoid unnecessary loss of the protein of interest. 
    2. Elute the protein using the Elution Buffer with a flow rate of 2 ml/min while fractionating to 1 ml samples, collect the eluate and save the fractions in separate tubes labeled ‘Elution fraction #’. An example of a typical elution chromatogram of FnCas12a purified by Histrap HP (5 ml) affinity purification is shown in Figure 4. 


      Figure 4. Representative elution chromatogram of FnCas12a purified by Histrap HP (5 ml) affinity purification. 75 ml of cell-free extract was loaded on the column. Elution fractions were 1 ml and the flow rate was set at 2 ml/min. Absorbance at 280 nm is expressed in milli-absorbance units for the A280 (blue) and A254 (red). Please note that the A254 is not very informative after niNTA purification, as at this stage, the sample is contaminated with various nucleic acids. The green line indicates the concentration of Elution Buffer (0% at the start of the chromatogram and 100% at the final stage of the chromatogram).

    3. Dilute 10 µl of the collected fractions with 4x Laemmli protein sample buffer, heat for 5 min at 95 °C and resolve the samples on SDS-PAGE gel to assess the purity of the sample.
    4. Combine the elution fractions in which the protein is present and check the absorbance at 280 nm to estimate the protein concentration. The extinction coefficient of FnCas12a is 145,820 M-1 cm-1. Typical yield at this step is ~25 mg per liter of expression culture**. 
      Note: **The yield is most likely overestimated at this point due to protein and nucleic acid contaminations.
    5. Add 2 ml (final 1 mM) of 1 M DTT stock and 4 ml of 0.5 M EDTA stock to 2 L of Dialysis Buffer before use. Increase the volume of the combined protein fractions to 25 ml using the Dialysis Buffer. Add 1 mg TEV per 100 mg of protein.
    6. Take a dialysis membrane with an MWCO of 12,400 and soak it in the Dialysis Buffer for 1 min. Use a clamp to close the dialysis membrane on one end to make a bag.
    7. Pipet the combined protein sample into the dialysis membrane bag and close the other end with another clamp. Dialyze the sample overnight at 4 °C with slow stirring against 2 L of the Dialysis Buffer.  

    Day 5. Purification of FnCas12a–Part II
    1. Transfer the protein sample from the dialysis membrane into a 50 ml tube and centrifuge it for 10 min at 4,500 x g at 4 °C to remove potential precipitated proteins.
    2. Dilute the sample 1:1 using Sample Dilution Buffer*.
    3. Using a peristaltic pump, wash the column with 3-5 column volumes of distilled water to remove the solution in which the column resin is stored.
    4. Using a peristaltic pump, equilibrate the Heparin FF column with at least 5 column volumes of IEX-A Buffer with a flow rate of 2 ml/min.
    5. Using a peristaltic pump, load the protein sample onto the Heparin FF column.
    6. Equilibrate the ÄKTA FPLC with IEX-A Buffer until the absorbance at 280 nm reaches a steady baseline.
    7. Transfer the Heparin FF column to the ÄKTA FPLC and wash it with 10 ml IEX-A Buffer at 2 ml/min. Collect the flow-through in appropriately labeled clean tubes.
    8. Elute the protein using a linear gradient from 0 to 50% IEX-B Buffer over 60 ml at a flow rate of 2 ml/min, collect the eluate as 1 ml fractions in appropriately labeled clean tubes. An example of a typical elution chromatogram of FnCas12a purified by Heparin FF (5 ml) affinity purification is shown in Figure 5. 


      Figure 5. Representative elution chromatogram of FnCas12a purified by Heparin FF (5 ml) affinity purification. 50 ml of sample was loaded. The flow rate was set at 2 ml/min and elution fractions were 1 ml. Absorbance is expressed in milli-absorbance units for the A280 (blue) and A254 (red). The green line indicates the concentration of IEX-B (0% at the start of the chromatogram with the concentration raising to 50% over 60 ml, and at 100% at the final stage of the chromatogram to wash the column).

    9. Dilute 10 µl of the collected fractions with 4x Laemmli protein sample buffer, heat for 5 min at 95 °C, and analyze on SDS-PAGE gel to assess the purity of the sample. An example of a Coomassie Brilliant Blue stained 10% SDS-PAGE gel on which FnCas12a Heparin FF elution fractions were resolved is shown in Figure 6.


      Figure 6. Representative Coomassie Brilliant Blue stained 12% SDS-PAGE gel on which FnCas12a Heparin FF elution fractions were resolved. M: PageRulerTM Plus Prestained Protein Ladder. Marker band sizes are indicated in kDa. IN: concentrated input sample of TEV protease-treated Histrap HP elution fractions after overnight dialysis. FT: Flow through from the column. Besides the large band formed by FnCas12a, other (contamination) bands can be observed. A9-C1: Elution fractions near the protein absorbance peak. The FnCas12a protein appears as a band with a size slightly larger than 130 kDa. In this case, fractions B14-C1 were combined.

    10. Combine the elution fractions containing pure protein (and as little contaminants as possible) and concentrate the sample by transferring the sample to Amicon Ultra-15 Centrifugal Filter Units with a membrane MWCO of 100 kD and centrifuging the samples at 4,500 x g at 4 °C until a final volume of < 1 ml is reached.
    11. Transfer the sample to an Eppendorf tube and centrifuge the sample for 2 min at maximum speed in a pre-cooled (4 °C) microcentrifuge to remove potential precipitation.
    12. Equilibrate a 2-ml injection loop and the HiLoad 16/600 Superdex 200 pg column with 10 ml and 240 ml SEC Buffer, respectively, on the ÄKTA FPLC.
    13. Load the protein concentrate on the HiLoad 16/600 Superdex 200 pg column using the 2-ml injection loop and resolve the sample on the column using SEC Buffer with a flow rate of 1 ml/min.
    14. Collect 1 ml fractions. An example of a typical elution chromatogram of FnCas12a purified by HiLoad 16/600 Superdex 200 pg column size exclusion purification is shown in Figure 7.


      Figure 7. Representative elution chromatogram of FnCas12a resolved on a HiLoad 16/600 Superdex 200 pg column. 1 ml of sample was loaded. Elution fractions were 1 ml and the flow rate was set at 1 ml/min. Absorbance is expressed in milli-absorbance units for the A280 (blue) and A254 (red).

    15. Dilute 10 µl of the collected fractions with 4x Laemmli protein sample buffer, heat for 5 min at 95 °C, and resolve on a 10% SDS-PAGE gel to assess the purity of the sample. An example of a Coomassie Brilliant Blue stained 10% SDS-PAGE gel on which FnCas12a SEC elution fractions were resolved is shown in Figure 8.


      Figure 8. Representative Coomassie Brilliant Blue stained 12% SDS-PAGE gel on which FnCas12a SEC elution fractions were resolved. M: PageRuler Plus Prestained Protein Ladder. Marker band sizes are indicated in kDa. IN: concentrated input sample. Besides the large band formed by FnCas12a, other (contamination) bands can be observed. C1-C12: Elution fractions near the protein absorbance peak. The FnCas12a protein appears as a band with a size slightly larger than 130 kDa. In this case, fractions C3-C9 were combined.

    16. Combine and concentrate the fractions that contain the pure protein and no (or a negligible amount of) contaminants.
    17. For analysis of purity and final protein yield, see the Data analysis section. The typical protein yield at this step is ~5-10 mg of FnCas12a per liter of E. coli expression culture. Expression and purification of AsCas12a or LbCas12a typically results in slightly higher Cas12a yields.
    18. Dilute the samples to a concentration suitable for subsequent experiments (e.g., 10 µM)** and aliquot the protein at a desired concentration and store at -80 °C.
      Notes:
      1. *For LbCas12a, dilute in a 2:1 ratio (protein sample: Dilution Buffer) due to the instability of LbCas12a at lower salt concentrations.
      2. **It is recommended to store the protein at ≥ 10 µM and dilute it to the right concentration only just before use. At lower concentrations, a relative high fraction of the protein can be lost due freezing/thawing and non-specific adsorption to the surface of the tube/container used for storage.

    Day 6. In vitro cleavage assay for confirming the activity of purified FnCas12a
    Notes:
    1. We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination.
    2. The reaction volume is typically 20 μl but can be scaled up as needed. Reactions should be assembled in nuclease-free 1.5 ml Eppendorf tubes or in 200 µl PCR (strip) tubes.
    3. Prepare a 1 µM crRNA solution by diluting the stock with nuclease-free water on ice.
    4. Prepare a 0.1 µM substrate plasmid or linear DNA solution by diluting the stock with nuclease-free water on ice.

    1. Prepare the following two-step reaction (at a molar ratio Cas12a:crRNA:substrate = 10:20:1) at room temperature:

    2. Mix thoroughly and pulse-spin in a micro-centrifuge.
    3. Incubate at 37 °C for 30 min.
    4. Add 1 µl of Proteinase K, mix thoroughly and pulse-spin in a micro-centrifuge.
    5. Incubate at room temperature for 10 min.
    6. Add 4 µl of 6x DNA loading dye.
    7. Resolve 20 µl of the sample on an 1% agarose gel pre-stained with SYBRTM Safe DNA Gel Stain.
    8. Visualize the gel using an imaging system equipped with an excitation source in the UV range or between 470-530 nm. An example of an in vitro cleavage assay using FnCas12a:crRNA and a linear DNA substrate is shown in Figure 9.


      Figure 9. A 2,774 bp linear target DNA substrate is cleaved by the FnCas12:crRNA complex, yielding products of 1,209 bp and 1,565 bp. M: GeneRuler 1 kb DNA Ladder.

Data analysis

Note: This section explains how to determine the yield and purity of your protein after the final step of the purification protocol (i.e., after size exclusion chromatography).

  1. Use a NanoDrop Spectrophotometer to measure the protein concentration. When using the NanoDrop software method ‘Protein A280’, both the absorption at 260 nm and the absorption at 280 nm are measured.
  2. The 260/280 ratio of the purified protein sample can be used to determine if the complex is free from nucleic acids. Typically, a pure protein sample has a 260/280 ratio of ~0.57. We typically achieve a 260/280 ratio between 0.54 and 0.61. Nucleic acid contamination rapidly increases the 260/280 ratio to above 1.
  3. Use the measured absorbance at 280 nm to determine the final protein concentration.
    1. When using the ‘Other protein (ε + MW)’ option of the NanoDrop software, provide the molar extinction coefficient of FnCas12a (144,330 M-1 cm-1) and its molecular weight (151 kDa). For AsCas12a, these values are 15,780 M-1 cm-1 and 156 kDa. For LbCas12a, these values are 181,690 M-1 cm-1 and 149 kDa.
    2. When using the ‘1 Abs = 1 mg/ml’ option of the NanoDrop software, use a correction factor to determine the real protein concentration. The correction factors for FnCas12a, AsCas12a, and LbCas12a are 0.951, 1.009 and 1.221, respectively. For example, if the NanoDrop measurement gives a protein concentration of 5.0 mg/ml of FnCas12a (assuming 1 Abs = 1 mg/ml), your real protein concentration is 5/0.951 = 5.26 mg/ml. To calculate the protein concentration in mM, use the following formula: protein concentration (in mg/ml)/molecular weight (in kDa; given above). For example, if you have a FnCas12a sample with 5.26 mg/ml protein, the protein concentration is 5.26/151 = 0.035 mM = 35 µM.

Recipes

  1. Media, antibiotics and stock solutions
    1. LB medium (1 L)
      1. Weigh out 10 g tryptone, 5 g yeast extract, and 10 g NaCl
      2. Fill up to 800 ml with demi-water
      3. Adjust pH to 7.5 with NaOH
      4. Fill up to 1 L with demi-water
      5. Autoclave at 121 °C for 30 min
      6. Store at room temperature
    2. 1,000x chloramphenicol stock (34 mg/ml stock)
      1. Weigh out 0.34 g chloramphenicol and dissolve it in 10 ml of 100% ethanol
      2. Pass through a 0.22 μm syringe filter
      3. Store at -20 °C
    3. 1,000x kanamycin stock (50 mg/ml stock)
      1. Weigh out 0.5 g kanamycin sulfate and dissolve it in 10 ml of sterile water
      2. Pass through a 0.22 μm syringe filter
      3. Store at -20 °C
    4. Glycerol stock (50% solution)
      1. Add 50 ml of 100% glycerol solution into a 250-ml bottle. When pipetting glycerol, use ethanol sterilized scissors to cut off the end of a pipette to make pipetting easier
      2. Add 50 ml demi-water
      3. Autoclave at 121 °C for 30 min
      4. Store at room temperature
    5. 1 M IPTG (IsoPropyl-1-Thio-β-D-Galactopyranoside)
      1. Weigh out 2.38 g of IPTG and dissolve in 10 ml of sterile water
      2. Pass through a 0.22 μm syringe filter
      3. Store at -20 °C
    6. 1 M DTT (Dithiothreitol) stock
      1. Weigh out 1.5 g DTT and dissolve it in 10 ml of sterile water
      2. Pass through a 0.22 μm syringe filter
      3. Store in the dark at -20 °C
    7. 0.5 M EDTA (Disodium Ethylene Diamine Tetra-Acetate) stock (pH 8.0)
      1. Weigh out 18.16 g of Na2EDTA·2H2O and dissolve in 80 ml of demi-water*
      2. Adjust to pH 8.0 with pellets of NaOH (~2 g of NaOH is required)
      3. Fill up to 100 ml with demi-water
      4. Sterilize by autoclaving Autoclave at 121 °C for 30 min
      5. Store at room temperature
        *Note: The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approximately 8.0 by the addition of NaOH.

  2. Buffers
    1. Lysis Buffer (1 L)
      1. Weigh out 29.22 g NaCl (final 500 mM), 2.42 g Tris (final 20 mM) and 0.68 g imidazole (final 10 mM) and dissolve in 900 ml of demi-water
      2. Adjust pH to 8.0 using HCl
      3. Fill up to 1 L with demi-water
      4. Filter using 0.22 µm membrane filter
      5. Store at 4 °C
    2. Wash Buffer (1 L)
      1. Weigh out 29.22 g NaCl (final 500 mM), 2.42 g Tris (final 20 mM) and 1.36 g imidazole (final 20 mM) and dissolve in 900 ml of demi-water
      2. Adjust pH to 8.0 using HCl
      3. Fill up to 1 L with demi-water
      4. Filter using 0.22 µm membrane filter
      5. Store at 4 °C
    3. Elution Buffer (1 L)
      1. Weigh out 29.22 g NaCl (final 500 mM), 2.42 g Tris (final 20 mM) and 17 g imidazole (final 250 mM) and dissolve in 900 ml of demi-water
      2. Adjust pH to 8.0 using HCl
      3. Fill up to 1 L with demi-water
      4. Filter using 0.22 µm membrane filter
      5. Store at 4 °C
    4. Dialysis Buffer (2 L)
      1. Weigh out 37.27 g KCl (final 250 mM) and 4.77 g HEPES (final 20 mM) and dissolve in 1,900 ml of demi-water
      2. Adjust pH to 8.0 using KOH
      3. Fill up to 2 L with demi-water
      4. Filter using 0.22 µm membrane filter
      5. Store at 4 °C
    5. Dilution Buffer (200 ml)
      1. Weigh out 0.24 g HEPES (final 10 mM) and dissolve in 100 ml of demi-water
      2. Adjust pH to 8.0 using KOH
      3. Fill up to 200 ml with demi-water
      4. Filter using 0.22 µm membrane filter
      5. Store at 4 °C
    6. IEX-A Buffer (1 L)
      1. Weigh out 11.18 g KCl (final 150 mM) and 4.77 g HEPES (final 20 mM) and dissolve in 900 ml of demi-water
      2. Adjust pH to 8.0 using KOH
      3. Fill up to 1 L with demi-water
      4. Filter using 0.22 µm membrane filter
      5. Store at 4 °C
    7. IEX-B Buffer (1 L)
      1. Weigh out 149.10 g KCl (final 2 M) and 4.77 g HEPES (final 20 mM) and dissolve in 900 ml of demi-water
      2. Adjust pH to 8.0 using KOH
      3. Fill up to 1 L with demi-water
      4. Filter using 0.22 µm membrane filter
      5. Store at 4 °C
    8. SEC Buffer (1 L)
      1. Weigh out 37.27 g KCl (final 500 mM), 4.77 g HEPES (final 20 mM) and dissolve in 900 ml of demi-water. Add 1 ml (final 1 mM) of 1 M DTT stock just before use
      2. Adjust pH to 8.0 using KOH
      3. Fill up to 1 L with demi-water
      4. Filter using 0.22 µm membrane filter
      5. Store at 4 °C
        Note: Setting the pH of the buffers at different temperatures will influence the final pH as the pH is temperature dependent. However, in our experience, the pH of the buffers can be set at 4 °C or at RT without having a significant impact on the purification procedure.
    9. 10x SDS-PAGE Electrophoresis Running Buffer (1 L)
      1. Weigh out 30.0 g Tris (final 250 mM), 144 g glycine (final 1920 mM), 10 g SDS [final 1% (w/v)] and dissolve in 900 ml of demi-water
      2. Fill up to 1 L with demi-water
    10. 10x Cas9 Nuclease Reaction Buffer (10 ml)
      1. Weight out 0.58 g NaCl (final 1 M), 0.1 g MgCl2·6H2O (final 50 mM), 0.476 HEPES (final 200 mM) and 3.72 mg EDTA (final 1 mM) and dissolve in 8 ml of nuclease-free water
      2. Adjust pH to 6.5 using HCl and fill up to 10 ml with nuclease-free water

Acknowledgments

This protocol is adapted from Swarts et al. (2017; Reference 26). This work was financially supported by the grant from the Netherlands Organization of Scientific Research (NWO) to J.v.d.O (NWO-TOP, 714.015.001.), by a Swiss National Science Foundation (SNSF) Project Grant to M.J. (SNSF 31003A_149393), and by fellowships of the European Molecular Biology Organization (EMBO) to D.C.S. (EMBO ALTF 179-2015 and EMBO aALTF 509-2017). The authors declare no conflicts of interest or competing interests.

References

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简介

该协议提供了分步说明(图1),用于在大肠杆菌中异源表达新西兰弗朗西斯菌弗朗西丝菌Cas12a(以前称为Cpf1)。 它还包括一个高纯度纯化方案,并简要介绍如何进行活性测定。 这些方案也可以用于其他Cas12a同系物的纯化,并且纯化的蛋白质可以用于随后的基因组编辑实验。

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图1.从大肠杆菌 异源表达和纯化<弗朗西斯弗朗西丝菌 Cas12a(FnCas12a)的活动时间表

【背景】原核CRISPR-Cas免疫系统通过使用CRISPR RNA(crRNA)作为外源DNA或RNA的序列特异性靶向的指导来提供针对病毒和质粒的保护(van der Oost等人,2014; Marraffini ,2015)。 1类CRISPR-Cas系统(包含I型,III型和IV型)通常形成多亚基蛋白-cRNA效应复合物,而2类系统(包含II型,V型和VI型)依赖于单个crRNA-引导的效应物核酸酶用于目标干扰(Mohanraju et al。 2016年)。

来自2类CRISPR-Cas系统的效应核酸酶已经成为用于基因组编辑和基因表达控制的有效且精确的工具(Mali et al。,2013; Doudna and Charpentier,2014; Hsu&lt; et al 。,2014)。被广泛使用的Cas9是II型系统的特征蛋白,它利用由crRNA和反式激活crRNA(tracrRNA)组成的双导向RNA结构进行目标识别(Deltcheva et al。,2011 )。对于基因组编辑目的,双引导RNA通常被成熟crRNA和tracrRNA的合成融合体替代,导致长的单分子引导RNA(sgRNA),其中单个RNA通过短接头序列融合(Jinek < 。,2012)。指导RNA的序列允许通过碱基配对与靶链结合互补DNA靶,而DNA的另一条链置换。在发现同源DNA靶标时,Cas9的HNH和RuvC核酸酶结构域分别介导靶标和置换链的切割(Jinek等人,2012; Karvelis等人 >,2013)。

最近,已在细菌基因组中鉴定了另一种具有独特特征的新型2类CRISPR-Cas核酸酶:Cas12a(也称为Cpf1)(Makarova和Koonin,2015; Zetsche等人,2015; Shmakov et al 。,2017)。 Cas12a利用单个crRNA指导进行DNA靶向;它不需要tracrRNA,与Cas9使用的嵌合单分子指导RNA(sgRNA)相比,产生更短的gRNA序列。虽然Cas9需要RNase III介导的pre-crRNA处理或sgRNA的单个表达以形成成熟的指导RNA,但Cas12a可以处理其自身的pre-crRNA。这种pre-crRNA加工活性允许在Cas12a介导的基因组编辑中进行简单复合(Wang等人,2017; Zetche等人,2017)。鉴于Cas9产生双链DNA断裂(DSBs),而Cas12a产生交错末端DSBs(Zetsche等人,2015年)。这样的突出可用于基于突出的克隆(Li等人,2016; Lei等人,2017)。此外,Cas9通常识别富含G的PAM序列,而迄今表征的所有Cas12a直向同源物识别富含T的PAM序列(Zetsche等人,2015)。总之,这些特征使Cas12a成为基因组编辑工具箱的重要补充。

已经成功地将Cas12a用于哺乳动物细胞中的基因组编辑应用(Zetche等人,2015; Kim等人,2016a),小鼠(Hur等人 。,2016; Kim et al。,2016b),rice(Endo em et al。,2016; Hu et al et al。 ,2017; Xu等人,2017),酵母(Verwaal等人,2017; Swiat等人,2017),斑马鱼(Moreno-Mateos等人,2017),微藻(Ferenczi等人,2017)和植物细胞(Zaidi >等人,2017; Kim等人,2017; Tang等人,2017)。与Cas9相比,Cas12a在人类细胞中的高效率和特异性以及较少的脱靶切割事件(Kleinstiver等人,2016)使Cas12a成为基因组编辑的稳健可靠的工具。

为了进行体外表征和结晶(Swarts等人,2017),从 Francisella novicida U112的Cas12a在 >大肠杆菌。 表达菌株大肠杆菌Rosetta TM 2(DE3)携带在IPTG诱导型lacUV5启动子控制下的染色体T7RNA聚合酶基因。 使用pET载体(Studier和Moffatt,1986; Rosenberg等人,1987; Studier等人,1990)表达cas12a基因 )用lacI控制的T7启动子进行。 在这里我们描述了控制表达和纯化FnCas12a所需的步骤。 该方案也可以用于表达和纯化来自酸性氨基球菌的Cas12a同系物。 和Lachnospiraceae细菌。

关键字:CRISPR-Cas, Cas12a, Cpf1, 蛋白质纯化

材料和试剂

注意:相同的材料和试剂可能被用作替代品。

  1. FnCas12a在E中的表达大肠杆菌Rosetta TM 2(DE3)
    1. 100-ml锥形瓶(DWK Life Sciences,DURAN,目录号:21 213 24)
    2. 2-L锥形瓶(DWK Life Sciences,DURAN,目录号:21 216 63)
    3. 5-L锥形瓶(DWK Life Sciences,DURAN?,目录号:21 216 73)
    4. 50-ml锥形离心管(Sigma-Aldrich,目录号:T2318-500EA)
    5. 2毫升螺旋顶管(康宁,目录号:430659)
    6. 带有密封封闭的Nalgene TM PPCO离心瓶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:3141-0500)或相当的500-ml离心瓶
    7. 移液器吸头(DeckWorks TM 标准移液吸头,Corning,目录号:4110; 4112; 4867)
    8. 10毫升注射器(BD,目录号:309604)
    9. 0.22μm注射器过滤器(Mdi,目录号:SYPL0601MNXX204)
    10. 250毫升瓶(Greiner Bio One International,目录号:227261)
    11. 大肠杆菌Rosetta TM 2(DE3)细胞(Merck,Novagen,目录号:71400)[编码在lacUV5控制下的T7RNA聚合酶基因] em>启动子]
    12. 质粒pDS015 * [pET His6 TEV LIC克隆载体(Addgene,目录号:29653)与F。 novicida U112 cas12a 基因插入片段与N端His标签融合;表达受lacI控制的T7启动子控制] *注:Acidaminococcus sp。也可以使用该表达将表达载体6His-MBP-TEV-huAsCpf1(Addgene,目录号:90095)和6His-MBP-TEV-huLbCpf1(Addgene,Invitrogen)的BV3L6 Cas12a(AsCas12a)和滑丝螺杆菌细菌ND2006 Cas12a(LbCas12a)目录号:90096)
    13. 胰蛋白胨(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:LP0042B)
    14. 酵母提取物(BD,Bacto TM,目录号:212720)
    15. 氯化钠(NaCl)(Fisher Scientific,目录号:S271-10)
    16. 氢氧化钠(NaOH)(Merck,EMD Millipore,目录号:106462)
    17. 乙醇(Fisher Scientific,目录号:BP2818500)
    18. 氯霉素(Fisher Scientific,目录号:BP904100)
    19. 卡那霉素硫酸盐(Thermo Fisher Scientific,目录号:11815024)
    20. 甘油(Fisher Scientific,目录号:BP229-4)
    21. IPTG(Fisher Scientific,产品目录号:BP1755-1)
    22. 琼脂(Acros Organics,目录号:400400050)
    23. LB培养基(见食谱)
    24. 1,000x氯霉素溶液(34 mg / ml)(见食谱)
    25. 1,000x卡那霉素溶液(50mg / ml)(见食谱)
    26. 1M IPTG(异丙基-1-硫代-β-D-吡喃半乳糖苷)(见食谱)
    27. 甘油库存(50%溶液)(见食谱)&nbsp;

  2. FnCas12a的纯化
    1. 5毫升HisTrap HP(GE Healthcare,目录号:17524701)
    2. 透析管,高保留无缝纤维素管,平均平宽23毫米(0.9英寸),截留分子量12,400,保留率99.99%(Sigma-Aldrich,目录号:D0405)
    3. 透析管夹(Sigma-Aldrich,目录号:Z371092)
    4. 5毫升HiTrap肝素HP(GE Healthcare,目录号:17040601)
    5. 具有Ultracel-100膜的Amicon Ultra-15离心过滤装置(Merck,EMD Millipore,目录号:UFC9100)
    6. HiLoad 16/600 Superdex 200 pg(GE Healthcare,产品目录号:28989335)
    7. Gosselin TM Round-Base 10-ml试管(Corning,Gosselin TM,目录号:TP10-01)或其他相当的级分收集管
    8. Nalgene TM Oak Ridge高速离心管(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:3114-0050)或相当的50-ml离心管
    9. 膜过滤器,混合纤维素酯(Merck,MF-Millipore,目录号:HAWP04700)
    10. 膜过滤器,混合纤维素酯(Merck,MF-Millipore,目录号:GSWP04700)
    11. 来自过夜培养的细胞沉淀,其中FnCas12a表达(来自程序A)
    12. 无EDTA的蛋白酶抑制剂混合物(Sigma-Aldrich,Roche Diagnostics,目录号:11873580001)
    13. 来自鸡蛋白的溶菌酶(Sigma-Aldrich,目录号:L6876-5G)
    14. β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
    15. TEV蛋白酶(Sigma-Aldrich,目录号:T4455)
    16. 12%Mini-PROTEAN TM TGX TM预制蛋白凝胶(Bio-Rad Laboratories,目录号:4561043)
    17. 用于SDS-PAGE的4×Laemmli蛋白样品缓冲液(Bio-Rad Laboratories,目录号:1610747)
    18. Bio-Safe TM Coomassie Stain(Bio-Rad Laboratories,目录号:1610786)
    19. PageRuler预染蛋白梯度,10至250 kDa(Thermo Fisher Scientific,Thermo Scientific TM,目录号:26619)
    20. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
    21. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E9884)
    22. 氯化钠(NaCl)(Fisher Scientific,目录号:S271-10)
    23. Tris(Thermo Fisher Scientific,Thermo Scientific TM,目录号:17926)
    24. 咪唑(Sigma-Aldrich,目录号:I0250)
    25. 盐酸(HCl)(Sigma-Aldrich,目录号:258148)
    26. 氯化钾(KCl)(Merck,EMD Millipore,目录号:104933)
    27. HEPES(Sigma-Aldrich,目录号:H3375)
    28. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:757551)
    29. 甘氨酸(Sigma-Aldrich,目录号:G8898)
    30. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
    31. 1 M DTT(二硫苏糖醇)库存(见食谱)
    32. 0.5M EDTA(乙二胺四乙酸二钠)储备液(pH8)(见食谱)
    33. 裂解缓冲液(见食谱)
    34. 洗涤缓冲液(见食谱)
    35. 洗脱缓冲液(见食谱)
    36. 透析缓冲液(见食谱)
    37. 稀释缓冲液(见食谱)
    38. IEX-A缓冲液(见食谱)
    39. IEX-B缓冲液(见食谱)
    40. SEC缓冲液(见食谱)
    41. 10x SDS-PAGE电泳运行缓冲液(见食谱)

  3. 使用纯化的Cas12a进行活性测定
    1. 纯化的Cas12a核酸酶(来自程序B)
    2. 无核酸酶的水
    3. 蛋白酶K,分子生物学级(新英格兰生物实验室,目录号:P8107S)
    4. 含有与靶DNA互补的靶向序列的crRNA
      注意:RNA可以作为脱盐RNA寡核苷酸订购,或者作为来自RNA合成公司如Sigma-Aldrich或IDT的PAGE纯化的RNA寡核苷酸订购。
    5. 含有靶序列和5'TTTN PAM序列的DNA底物
      注:底物DNA可以是环状或线性化的质粒,PCR产物或合成的寡核苷酸)。举例来说,活性测定中使用的DNA底物和crRNA如图2所示。


      图2. Cas12a crRNA-DNA靶向复合物的示意图预期的切割位点用红色箭头表示。

    6. GeneRuler 1 kb DNA Ladder(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:SM0311)或同等产品
    7. DNA凝胶负载染料[例如,6x DNA加载染料(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0611)]
    8. InvitrogenTM SYBR TM Safe DNA凝胶染色(Thermo Fisher Scientific,Thermo Scientific TM,目录号:S33102)
    9. 氯化钠(NaCl)(Fisher Scientific,目录号:S271-10)
    10. 氯化镁六水合物(MgCl 2·6H 2 O)
    11. HEPES(Sigma-Aldrich,目录号:H3375)
    12. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E9884)
    13. 盐酸(HCl)(Sigma-Aldrich,目录号:258148)
    14. 10x核酸酶反应缓冲液(见食谱)

设备

注意:可以使用等效设备。

  1. FnCas12a在E中的表达大肠杆菌Rosetta TM 2
    1. 移液器(Corning,型号:Lambda TM Plus单通道移液器,目录号:4070; 4074; 4075)
    2. 新不伦瑞克TM Innova 42培养箱(Eppendorf,New Brunswick TM,型号:Innova 42,目录号:M1335-0002)或可以设置在37°C的等效培养箱。
    3. Sorvall LYNX 4000超速离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Sorvall LYNX 4000,目录号:75006580)或可以冷却至4°C的等同离心机并且可以执行高达6,000 xg
    4. 新不伦瑞克TM Innova 44 / 44R(Eppendorf,New Brunswick TM,型号:Innova 44 / 44R ,产品目录号:M1282-0002)或任何同等的振荡培养箱,温度可以设定在37°C和18°C。
    5. 细胞密度计(GE Healthcare,型号:Ultrospec TM 10,目录号:80-2116-30)或可以测量600nm悬浮液中细胞密度的等效分光光度计。
    6. 冰水浴(水和冰混合)

  2. FnCas12a的纯化
    1. 带有VS 70 T Sonotrode(Bandelin)或同等超声波均质器/超声波仪或法国压力室(French Press)的SONOPULS HD(Bandelin electronic,型号:HD 3200)用于细胞裂解
    2. 带有5 ml HisTrap HP(GE Healthcare,型号:蠕动泵P-1,产品目录号:18111091)的连接器的蠕动泵P-1或同等蠕动泵

    3. 用于蠕动泵P-1(GE Healthcare,目录号:11300082)的管道连接器
    4. ÄKTApurifier10 FPLC系统(GE Healthcare,型号:ÄKTApurifier10,产品目录号:28406264)或同等FPLC系统
    5. Sorvall LYNX 4000超速离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Sorvall LYNX 4000,目录号:75006580)或可以冷却至4°C的等同离心机并且可以执行高达30,000 x g
    6. pH计(QiS,型号:B210)
    7. 用于过滤的过滤器支架组件(Merck,产品目录号:XX1014700或Nalgene,Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:DS0320-2545)或等效的过滤器支架组件。
    8. 膜片式真空泵LABOPORT N 820(ABM van Zijl B.V,目录号:ABMK N8203FT18)或等效真空泵
    9. Nanodrop(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 2000,目录号:ND-2000)
    10. Mini-PROTEAN Tetra细胞(Bio-Rad Laboratories,型号:Mini-PROTEAN Tetra Cell,目录号:1658004EDU)或等效的垂直电泳系统。
    11. 爱普生Perfection V850 Pro扫描仪(Epson,型号:Perfection V850 Pro)或适用于SDS-PAGE凝胶成像的等价扫描仪或成像仪。

  3. 使用纯化的Cas12a进行活性测定
    1. Eppendorf TM 5424微量离心机(Eppendorf,型号:5424,目录号:022620498)
    2. MUPID One水平电泳系统(Bulldog Bio,产品目录号:MU2)或等效的水平电泳系统
    3. G:BOX F3(Syngene,型号:G:BOX F3,目录号:05-GBOX-F3)gel doc系统或相当的DNA琼脂糖凝胶成像设备

程序

  1. E的转换。用含有pDS015质粒的大肠杆菌Rosetta TM 2(DE3)和制备甘油贮存液
    1. 将1ng pDS015质粒DNA直接加入到50μl化学感受态E中。大肠杆菌Rosetta TM 2(DE3)细胞。
    2. 轻轻搅拌以混合并将管置于冰上5分钟。
    3. 在42°C水浴中将管子加热30秒;不要动摇。
    4. 立即将试管置于冰上2分钟。
    5. 将250μl室温无菌SOC培养基(由制造商提供)加入管中。
      在37℃下孵育,同时以250rpm振荡60分钟。
    6. 将5-50μl转化混合物分别涂布在含有34μg/ ml和50μg/ ml氯霉素和卡那霉素的LB琼脂平板上。如果电镀少于25μl的转化,我们建议在电镀前向转化混合物中加入50μl无菌SOC培养基以促进LB琼脂平板表面上的菌落分布。
    7. 在37℃孵育LB琼脂平板过夜。
    8. 第二天,使用无菌枪头从转化平板上挑选单个菌落并接种10ml LB在50ml管中。
    9. 在37℃振荡培养箱中以160rpm振荡孵育50ml试管过夜。
    10. 第二天,在500毫升50%无菌甘油中加入500微升过夜培养液,置于2毫升螺旋管或冷冻管中,轻轻混合。
    11. 将甘油储存在-80°C以备将来使用。

  2. E中大量表达FnCas12a。大肠杆菌Rosetta TM 2(DE3)
    第1天。培养基,缓冲液和单菌落的制备
    1. 准备20毫升的LB在100毫升锥形瓶(用于开始过夜培养)(配方A1)。
    2. 准备三个5-L锥形瓶,每个包含1.5L的LB培养基(用于大规模繁殖和蛋白质纯化)(配方A1)。
    3. 准备抗生素和储备溶液(配方A2和A3)。
    4. 准备净化所需的缓冲液(配方B1-B8)。
    5. 排出甘油库存的E。在含有50μg/ ml卡那霉素和34μg/ ml氯霉素的LB琼脂平板上用pDS015转化的大肠杆菌Rosetta TM 2(DE3)。
    6. 在37℃培养箱中培养LB琼脂平板过夜。

    第2天。隔夜文化准备
    1. 将20μl50mg / ml卡那霉素储备溶液和20μl34mg / ml氯霉素储备溶液加入到来自第1天的100ml锥形瓶中的20ml高压灭菌的LB培养基中。
    2. 使用无菌吸头,选择一个单一的E菌落。用来自第1天的LB琼脂平板的pDS015转化的大肠杆菌Rosetta TM 2(DE3)。
    3. 使用菌落接种含有卡那霉素和氯霉素的培养基。

    4. 使用棉塞松散地关闭100毫升的锥形瓶
    5. 在振荡培养箱(设定为160rpm)中37℃孵育细菌培养物16-20小时。

    第3天3.过度表达FnCas12a的细胞大规模扩增
    1. 取三个高压灭菌的5升锥形瓶*,每个瓶含有来自第1天的1.5L LB培养基。
    2. 向每个烧瓶中加入1.5毫升50毫克/毫升的氯霉素溶液。
    3. 向每个烧瓶中加入1.5ml 34mg / ml卡那霉素溶液。
    4. 向每个烧瓶中加入15ml在第2天制备的过夜培养物。

    5. 在摇床中以160转/分的速度在37°C孵育培养瓶
    6. 每半小时监测培养物的OD 600nm。一旦达到0.5-0.6的OD 600nm(通常需要约3-4小时),将含有培养物的锥形瓶转移到冰水浴中并孵育(冷冲击)以用于15分钟。这一步减缓了E的代谢。并触发表达可能有助于FnCas12a在表达过程中折叠的冷休克蛋白。
    7. 向每个培养瓶中加入200μl过滤灭菌的1M IPTG溶液至培养物以诱导FnCas12a的表达。
    8. 将培养物转移到18℃摇动培养箱(设定为120rpm)过夜表达(约16小时)。
      注意:
      1. *也可以表达较小体积的细胞培养物(例如,在2-L锥形瓶中的单个1.5L培养物或一个或多个750ml培养物)
      2. **当使用带挡板的锥形瓶时,将振荡培养箱的速度降低至120rpm以防止形成泡沫。&nbsp;

    第四天 - 第一部分。过度表达FnCas12a的细胞的大规模增殖(续)
    1. 将第3天的过夜培养物转移到离心瓶中。
    2. 通过在4℃以6,000×gg离心培养物15分钟来收获细胞。
    3. 弃去上清液并将沉淀的细胞储存在-20°C(一周内用于最佳纯化)或-80°C(用于长期储存)或直接进行纯化。预期产量为每升细胞培养物约5克细胞沉淀。

    第4天 - 第二部分。纯化FnCas12a-I部分
    1. 如果继续使用冷冻细胞沉淀物,将细胞沉淀物从冰上第4天 - 第I部分解冻30-60分钟。如果在蛋白质表达后直接进行,请跳过此步骤。
      注意:所有后续步骤应在冰上或4°C下进行。
    2. 在裂解缓冲液(每克细胞沉淀约2.5-5ml裂解缓冲液)中重悬全部细胞沉淀。
    3. 每50 ml添加1片cOmplete TM蛋白酶抑制剂。
    4. 加入溶菌酶至最终浓度为1mg / ml。
    5. 在冰上孵育30分钟。
    6. 如果使用French Press进行细胞裂解:在溶菌酶处理后,将细胞悬浮液通过French Press在16,000 psi下两次。
    7. 如果使用超声处理进行细胞裂解:在溶菌酶处理后,通过使用具有适当尖端的超声波仪和适用于裂解大体积细胞悬液的方案来裂解细胞悬液。对于我们的设置(Bandelin SONOPULS HD with VS 70 T tip),我们使用以下设置:10分钟总时间,1秒开,0.7秒关和20%振幅。如图3所示,细胞悬液在裂解后通常具有淡褐色。
      注意:使用法国压力室或超声波仪可获得大致相同的产量-FnCas12a非常稳定,在超声处理过程中很少或不会有蛋白质丢失。但请记住,超声处理通常不适合大量使用,因此应适用于大量细胞悬液裂解的协议。


      图3.裂解后,细胞悬液呈棕褐色,粘稠性较低

    8. 将溶胞产物倒入(a)离心管中,并在30000gxg离心45分钟,4℃。
    9. 将上清液转移到(a)干净的50ml管中。这是'无细胞提取物'。
      注:可将样品溶解的细胞沉淀物储存在4°C下,通过SDS-PAGE分析后分析FnCas12a的存在以评估其溶解度并确定细胞溶解是否成功。 br />
    10. 将无细胞提取液通过0.22μm膜过滤器,并将滤液保存在无菌管中。
    11. 使用蠕动泵,使用3-5个柱体积的蒸馏水清洗HisTrap HP色谱柱,以除去储存树脂的溶液。
    12. 使用蠕动泵,用至少5个柱体积的裂解缓冲液平衡HisTrap HP,流速为2 ml / min。
    13. 使用蠕动泵*,将过滤后的无细胞提取物以1 ml / min的流速通过HisTrap HP色谱柱,并在(a)标记为“流过”的50 ml管中收集流通液* *。
      注意:
      1. *对于样品加载,可以使用超级循环代替蠕动泵。
      2. **流通样品可以保存在4°C,后来通过SDS-PAGE分析分析FnCas12a的存在以确定蛋白质是否与柱结合(如果蛋白质的大部分留在流通中,再生或更换柱子)。在某些情况下,高量的蛋白质可能会使色谱柱饱和。为了回收流通过程中的蛋白质,可以将流通(重新)加载到另一个或再生的HisTrap HP色谱柱上。&nbsp;

      下一步色谱步骤使用ÄKTAFPLC系统进行。
    14. 用Wash Buffer平衡ÄKTAFPLC系统,直至280 nm处的吸光度达到稳定基线。将HisTrap HP色谱柱转移到ÄKTAFPLC上,使用洗涤缓冲液以2 ml / min的流速洗脱色谱柱10-15倍,或直到280 nm处的吸光度用洗涤缓冲液接近**稳定。
      收集第一批3-4个10-ml洗涤级分*,放入单独的标记为“穿透#”的试管中。
      备注:
      1. 可以保存并在稍后使用SDS-PAGE分析检查FnCas12a的存在以确定它在洗涤过程中是否从柱上洗脱出来。
      2. 即使在低浓度下,洗涤缓冲液中的咪唑也可以除去少量感兴趣的蛋白质。因此,开始洗脱蛋白质时,280 nm处的吸光度接近稳定,以避免不必要的目标蛋白质损失。&nbsp;
    15. 使用洗脱缓冲液以2 ml / min的流速洗脱蛋白质,同时分馏至1 ml样品,收集洗脱液并将洗脱液保存在标有“洗脱级分#”的单独试管中。图4显示了通过Histrap HP(5ml)亲和纯化纯化的FnCas12a的典型洗脱色谱图的实例。&nbsp;


      图4.通过Histrap HP(5ml)亲和纯化纯化的FnCas12a的代表性洗脱色谱图将75ml无细胞提取物加载到柱上。洗脱级分为1ml,流速设定为2ml / min。 280nm处的吸光度以Δ280(蓝色)和Δ254(红色)的毫体吸光度单位表示。请注意,在niNTA纯化后,A254不具有很多信息,因为在这个阶段,样品被各种核酸污染。绿线表示洗脱缓冲液的浓度(在色谱图开始时为0%,在色谱图的最后阶段为100%)。

    16. 用4x Laemmli蛋白样品缓冲液稀释10μl收集的级分,95°C加热5 min,然后在SDS-PAGE凝胶上分离样品以评估样品的纯度。
    17. 将存在蛋白质的洗脱组分合并,并检查280nm处的吸光度以估计蛋白质浓度。 FnCas12a的消光系数为145,820 M -1 -1 cm -1。此步骤的典型产量为〜25 mg /升表达培养**。
    18. 在使用前向2L透析缓冲液中加入2ml(最终1mM)1M DTT原液和4ml 0.5M EDTA原液。使用透析缓冲液将混合的蛋白质组分的体积增加至25ml。每100毫克蛋白添加1毫克TEV。
    19. 以MWCO为12,400的透析膜并将其浸入透析缓冲液中1分钟。使用夹子将透析膜的一端封闭,制成袋子。
    20. 将组合的蛋白质样品吸入透析膜袋并用另一个夹钳关闭另一端。在4℃缓慢搅拌下透析2小时的透析缓冲液。
      注意:
      **由于蛋白质和核酸污染,此时产量很可能被高估。&nbsp;

    第5天。FnCas12a-Part II的纯化
    1. 将来自透析膜的蛋白质样品转移至50ml管中,并在4℃下以4,500×gg离心10分钟以除去潜在的沉淀蛋白质。

    2. 使用样本稀释缓冲液* 1:1稀释样本
    3. 使用蠕动泵,用3-5个柱体积的蒸馏水清洗色谱柱,以去除色谱柱树脂储存的溶液。
    4. 使用蠕动泵,用至少5个柱体积的IEX-A缓冲液平衡肝素FF柱,流速为2 ml / min。
    5. 使用蠕动泵将蛋白质样品加载到肝素FF柱上。
    6. 用IEX-A缓冲液平衡ÄKTAFPLC,直到280 nm处的吸光度达到稳定基线。
    7. 将肝素FF柱转移至ÄKTAFPLC,并用2 ml / min的10 ml IEX-A缓冲液清洗。用适当标记的清洁管收集流通物。
    8. 用流速为2 ml / min的0至50%IEX-B缓冲液以60 ml的线性梯度洗脱蛋白质,将洗脱液以1 ml流分收集在适当标记的干净管中。图5显示了通过肝素FF(5ml)亲和纯化纯化的FnCas12a的典型洗脱色谱图的实例。&nbsp;


      图5.通过肝素FF(5ml)亲和纯化纯化的FnCas12a的代表性洗脱色谱图。加载50ml样品。流速设定为2ml / min,洗脱级分为1ml。 A 280(蓝色)和A 254(红色)的吸光度用毫安吸光度单位表示。绿线表示IEX-B的浓度(在色谱开始时为0%,浓度在60毫升时上升到50%,在色谱图的最后阶段,浓度上升到100%以清洗色谱柱) >
    9. 用4x Laemmli蛋白样品缓冲液稀释10μl收集的级分,95°C加热5 min,然后在SDS-PAGE凝胶上分析以评估样品的纯度。考马斯亮蓝染色的10%SDS-PAGE凝胶的实例如图6所示.FnCas12a肝素FF洗脱级分在其上被分辨。


      图6.代表性的考马斯亮蓝染色12%SDS-PAGE凝胶,FnCas12a肝素FF洗脱组分被分辨。M:PageRuler TM Plus Prestained Protein Ladder。标记带的大小以kDa表示。 IN:过夜透析后浓缩的TEV蛋白酶处理的Histrap HP洗脱级分的输入样品。 FT:从柱子流出。除了由FnCas12a形成的大带外,还可以观察到其他(污染)带。 A9-C1:蛋白质吸收峰附近的洗脱级分。 FnCas12a蛋白表现为尺寸略大于130kDa的条带。在这种情况下,馏分B14-C1被合并。

    10. 将含有纯蛋白质(尽可能少的污染物)的洗脱级分组合并通过将样品转移至具有100kD膜MWCO的Amicon Ultra-15离心过滤器单元并将样品在4,500xg /在4℃下,直至最终体积为&lt;达到1毫升。
    11. 将样品转移至Eppendorf管中,并在预冷的(4℃)微量离心机中以最大速度离心样品2分钟以去除潜在的沉淀。
    12. 在ÄKTAFPLC上分别用10 ml和240 ml SEC缓冲液平衡2 ml注射环和HiLoad 16/600 Superdex 200 pg柱。
    13. 使用2ml注射环将蛋白浓缩物加载到HiLoad 16/600 Superdex 200 pg柱上,并使用SEC缓冲液以1ml / min的流速将样品溶解在柱上。
    14. 收集1毫升的馏分。 HiLoad 16/600 Superdex 200 pg柱尺寸排阻纯化纯化的FnCas12a的典型洗脱色谱图示例如图7所示。


      图7.在HiLoad 16/600 Superdex 200 pg柱上解析的FnCas12a的代表性洗脱色谱图。加载1ml样品。洗脱级分为1ml,流速设定为1ml /分钟。 A 280(蓝色)和A 254(红色)的吸光度用毫安吸光度单位表示。

    15. 用4x Laemmli蛋白样品缓冲液稀释10μl收集的级分,在95°C加热5 min,然后在10%SDS-PAGE凝胶上分离,以评估样品的纯度。考马斯亮蓝染色的10%SDS-PAGE凝胶的实例显示在图8中.FnCas12a SEC洗脱级分在其上被分辨。


      图8.代表考马斯亮蓝染色的12%SDS-PAGE凝胶,其上分辨FnCas12a SEC洗脱级分。M:PageRuler Plus预染蛋白梯级。标记带的大小以kDa表示。 IN:浓缩的输入样本。除了由FnCas12a形成的大带外,还可以观察到其他(污染)带。 C1-C12:靠近蛋白质吸收峰的洗脱部分。 FnCas12a蛋白表现为尺寸略大于130kDa的条带。在这种情况下,馏分C3-C9被合并。

    16. 合并和浓缩含有纯蛋白质且不含(或可忽略量)污染物的馏分。
    17. 有关纯度和最终蛋白质产量的分析,请参阅数据分析部分。此步骤中典型的蛋白质产量为每升E-E约5-10mg FnCas12a。大肠杆菌表达培养。 AsCas12a或LbCas12a的表达和纯化通常会导致Cas12a产量稍高。
    18. 将样品稀释至适合后续实验的浓度(例如,10μM)**并将蛋白质等分至所需浓度并储存于-80℃。
      注意:
      1. *对于LbCas12a,由于在较低盐浓度下LbCas12a的不稳定性,以2:1比例(蛋白质样品:稀释缓冲液)稀释。
      2. **建议将蛋白质储存在≥10μM,并在使用前将其稀释到合适的浓度。在较低浓度时,由于冷冻/解冻和用于储存的管/容器表面的非特异性吸附,蛋白质的相对高比例可能会丢失。

    第6天:用于确认纯化的FnCas12a活性的体外切割试验
    注意:
    1. 我们强烈建议戴上手套并使用无核酸酶的试管和试剂以避免RNase污染。
    2. 反应体积通常为20μl,但可根据需要放大。反应应在无核酸酶1.5 ml Eppendorf管或200μlPCR(带)管中组装。
    3. 通过在冰上用无核酸酶的水稀释原液制备1μMcrRNA溶液。
    4. 通过在冰上用无核酸酶的水稀释原液制备0.1μM底物质粒或线性DNA溶液。

    1. 在室温下制备以下两步反应(摩尔比Cas12a:crRNA:底物= 10:20:1):

    2. 充分混合并在微型离心机中进行脉冲旋转。
    3. 37°C孵育30分钟。
    4. 加1μl蛋白酶K,充分混合并在微型离心机中脉冲旋转。
    5. 在室温下孵育10分钟。
    6. 加入4μl6x DNA上样染料。
    7. 将样品溶解在1%琼脂糖凝胶上,用SYBR TM安全DNA凝胶染色剂预先染色。
    8. 使用配备有紫外线范围或470-530纳米之间的激发源的成像系统使凝胶可视化。图9中显示了使用FnCas12a:crRNA和线性DNA底物的体外剪切测定的实例。


    图9. 2,774bp线性靶DNA底物被FnCas12:crRNA复合物切割,产生1,209bp和1,565bp的产物。M:GeneRuler 1kb DNA Ladder。

数据分析

注意:本节解释了如何在纯化方案的最后一步(即尺寸排阻色谱法)之后确定蛋白质的产量和纯度。

  1. 使用NanoDrop分光光度计测量蛋白质浓度。当使用NanoDrop软件方法'蛋白A 280'时,测量260nm处的吸收和280nm处的吸收。
  2. 纯化的蛋白质样品的260/280比例可用于确定复合物是否不含核酸。通常,纯蛋白质样品具有〜0.57的260/280比率。我们通常在0.54和0.61之间达到260/280的比率。核酸污染迅速将260/280比率提高到1以上。
  3. 使用280nm处测得的吸光度来确定最终的蛋白质浓度。
    1. 当使用NanoDrop软件的'其他蛋白质(ε+ MW)'选项时,提供FnCas12a的摩尔消光系数(144,330 M -1 -1cm -1 -1)和它的分子量(151kDa)。对于AsCas12a,这些值分别为15,780 M -1 -1 cm -1和156 kDa。对于LbCas12a,这些值为181,690 M -1 -1cm -1和149 kDa。
    2. 当使用NanoDrop软件的'1 Abs = 1 mg / ml'选项时,使用校正因子来确定真正的蛋白质浓度。 FnCas12a,AsCas12a和LbCas12a的校正因子分别为0.951,1.009和1.221。例如,如果NanoDrop测量结果的蛋白质浓度为5.0 mg / ml FnCas12a(假设1 Abs = 1 mg / ml),则您的实际蛋白质浓度为5 / 0.951 = 5.26 mg / ml。为了计算以mM为单位的蛋白质浓度,使用以下公式:蛋白质浓度(以mg / ml计)/分子量(以kDa计;如上所述)。例如,如果您有含有5.26 mg / ml蛋白质的FnCas12a样品,则蛋白质浓度为5.26 / 151 = 0.035 mM = 35μM。

食谱

  1. 媒体,抗生素和储备溶液
    1. LB培养基(1L)
      1. 称出10克胰蛋白胨,5克酵母提取物和10克NaCl。

      2. 使用半水补充至800毫升
      3. 用NaOH调节pH至7.5

      4. 使用半水加注至1升
      5. 在121°C高压灭菌30分钟
      6. 在室温下储存
    2. 1,000x氯霉素原液(34mg / ml原液)
      1. 称出0.34克氯霉素,并将其溶解于10毫升100%乙醇中。
      2. 穿过0.22μm注射器过滤器
      3. 在-20°C储存
    3. 1,000x卡那霉素原液(50mg / ml原液)
      1. 称取0.5 g硫酸卡那霉素,并将其溶解于10 ml无菌水中。
      2. 穿过0.22μm注射器过滤器
      3. 在-20°C储存
    4. 甘油储备(50%溶液)
      1. 将50毫升100%甘油溶液加入250毫升瓶中。当移液甘油时,使用乙醇无菌剪刀切断移液器的末端以使移液更容易
      2. 添加50毫升半水
      3. 在121°C高压灭菌30分钟
      4. 在室温下储存
    5. 1M IPTG(异丙基-1-硫代-β-D-吡喃半乳糖苷)
      1. 称出2.38克IPTG并溶解于10毫升无菌水中
      2. 穿过0.22μm注射器过滤器
      3. 在-20°C储存
    6. 1 M DTT(二硫苏糖醇)库存
      1. 称取1.5克DTT,并将其溶解于10毫升无菌水中。
      2. 穿过0.22μm注射器过滤器

      3. 在-20°C黑暗处储存
    7. 0.5M EDTA(乙二胺四乙酸二钠)储备液(pH8.0)
      1. 称取18.16g Na 2 EDTA·2H 2 O并溶于80ml去离子水中。
      2. 用氢氧化钠颗粒调整至pH8.0(需要约2克NaOH)


      3. 使用半水补足至100毫升
      4. 通过高压灭菌高压灭菌器在121°C灭菌30分钟
      5. 在室温下储存
        *注:EDTA的二钠盐不会进入溶液,直到通过加入NaOH将溶液的pH调节至约8.0。

  2. 缓冲区
    1. 裂解缓冲液(1 L)
      1. 称出29.22g NaCl(最终500mM),2.42g Tris(最终20mM)和0.68g咪唑(最终10mM)并溶解于900ml去离子水中。
      2. 使用HCl将pH值调节至8.0

      3. 使用半水加注至1升
      4. 过滤器使用0.22微米膜过滤器
      5. 在4°C储存
    2. 清洗缓冲液(1 L)
      1. 称出29.22g NaCl(最终500mM),2.42g Tris(最终20mM)和1.36g咪唑(最终20mM),并溶解于900ml去离子水中。
      2. 使用HCl将pH值调节至8.0

      3. 使用半水加注至1升
      4. 过滤器使用0.22微米膜过滤器
      5. 在4°C储存
    3. 洗脱缓冲液(1L)
      1. 称出29.22g NaCl(最终500mM),2.42g Tris(最终20mM)和17g咪唑(最终250mM)并溶解于900ml去离子水中。
      2. 使用HCl将pH值调节至8.0

      3. 使用半水加注至1升
      4. 过滤器使用0.22微米膜过滤器
      5. 在4°C储存
    4. 透析缓冲液(2 L)
      1. 称取37.27克KCl(最终250毫摩尔)和4.77克HEPES(最终20毫摩尔)并溶解于1,900毫升去离子水中。
      2. 使用KOH调节pH值至8.0

      3. 使用半水加注至2升
      4. 过滤器使用0.22微米膜过滤器
      5. 在4°C储存
    5. 稀释缓冲液(200ml)
      1. 称取0.24 g HEPES(最终10 mM)并溶于100 ml去离子水中
      2. 使用KOH调节pH值至8.0


      3. 使用半水补足200毫升
      4. 过滤器使用0.22微米膜过滤器
      5. 在4°C储存
    6. IEX-A缓冲液(1L)
      1. 称出11.18克KCl(最终150毫摩尔)和4.77克HEPES(最后20毫摩尔)并溶解在900毫升去离子水中。
      2. 使用KOH调节pH值至8.0

      3. 使用半水加注至1升
      4. 过滤器使用0.22微米膜过滤器
      5. 在4°C储存
    7. IEX-B缓冲液(1 L)
      1. 称取149.10g KCl(最终2M)和4.77g HEPES(最终20mM)并溶于900ml去离子水中。
      2. 使用KOH调节pH值至8.0

      3. 使用半水加注至1升
      4. 过滤器使用0.22微米膜过滤器
      5. 在4°C储存
    8. SEC缓冲液(1 L)
      1. 称出37.27g KCl(最终500mM),4.77g HEPES(最终20mM)并溶解于900ml去离子水中。
        使用前添加1 ml(最终1 mM)1 M DTT原液
      2. 使用KOH调节pH值至8.0

      3. 使用半水加注至1升
      4. 过滤器使用0.22微米膜过滤器
      5. 在4°C储存
        注意:将pH值取决于温度时,将缓冲液的pH值设置为不同的温度会影响最终的pH值。然而,根据我们的经验,缓冲液的pH值可以设定在4°C或RT下,而不会对纯化过程产生重大影响。
    9. 10x SDS-PAGE电泳运行缓冲液(1L)
      1. 称取30.0g Tris(终浓度250mM),144g甘氨酸(终浓度为1920mM),10g SDS [终浓度1%(w / v)],并溶解于900ml去离子水中。

      2. 使用半水加注至1升
    10. 10x Cas9核酸酶反应缓冲液(10ml)
      1. 称取0.58g NaCl(最终1M),0.1g MgCl 2·6H 2 O(最终50mM),0.476HEPES(最终200mM)和3.72mg EDTA (最终1mM)并溶解在8ml无核酸酶的水中。
      2. 使用HCl将pH调节至6.5并用无核酸酶的水填充至10ml

致谢

该协议改编自Swarts等人(2017;参考文献26)。这项工作得到荷兰科学研究组织(NWO)对JvdO(NWO-TOP,714.015.001。)的资助,由瑞士国家科学基金会(SNSF)项目资助给MJ(SNSF 31003A_149393)由欧洲分子生物学组织(EMBO)向DCS提供奖学金(EMBO ALTF 179-2015和EMBO aALTF 509-2017)。作者声明不存在利益冲突或利益冲突。

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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mohanraju, P., Oost, J. v., Jinek, M. and Swarts, D. C. (2018). Heterologous Expression and Purification of the CRISPR-Cas12a/Cpf1 Protein. Bio-protocol 8(9): e2842. DOI: 10.21769/BioProtoc.2842.
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