A One-Step Method for Efficient Purification of Functional Cas9 Protein
一步法高效纯化具有功能活性的 Cas9 蛋白
(*contributed equally to this work) 发布: 2026年02月05日第16卷第3期 DOI: 10.21769/BioProtoc.5594 浏览次数: 157
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Sep 2025
Abstract
The CRISPR/Cas9 system is a cornerstone technology in genome editing. Delivery of pre-assembled Cas9 ribonucleoprotein (RNP) complexes exhibits distinct advantages, including reduced off-target effects and lower immunogenicity. Conventional methods for purifying Cas9 protein typically involve multi-step chromatography and the cleavage of fusion tag, which are time-consuming and result in diminished yields. In this study, we present a simplified, one-step purification strategy for functional Streptococcus pyogenes Cas9 (SpCas9) using the ubiquitin (Ub) fusion system in Escherichia coli. The N-terminal Ub fusion not only improves protein solubility but also facilitates high-yield production of the His-Ub-Cas9 fusion protein. Importantly, the Ub tag does not require proteolytic removal during purification, allowing direct one-step purification of the fusion protein via nickel-affinity chromatography. The purified His-Ub-Cas9 retains robust DNA cleavage activity in vivo, as validated in zebrafish embryos. This protocol greatly simplifies the production of functional Cas9 protein, facilitating its broad application in genome editing.
Key features
• The Ub fusion system enables single-step purification of Cas9 in E. coli using Ni-NTA chromatography, eliminating the protease cleavage step.
• This method yields over 8 mg/L of high purity (>95%), functional Cas9 protein, suitable for direct use in RNP complex assembly.
• The purified His-Ub-Cas9 maintains high genome editing activity in vivo, as demonstrated in zebrafish embryos.
Keywords: SpCas9 (SpCas9)
Protein purification (蛋白纯化)
Ubiquitin-fusion (泛素融合)
Affinity chromatography (亲和层析)
Gene editing (基因编辑)
Background
The CRISPR-Cas9 system has revolutionized genome editing by enabling precise, programmable modifications of DNA sequences across diverse biological systems. Owing to its simplicity, efficiency, and versatility, the CRISPR-Cas9 system has become an essential tool for basic scientific research, biotechnology applications, and the advancement of therapeutic strategies [1]. Although plasmid- and mRNA-based delivery methods are commonly used, Cas9 ribonucleoprotein (RNP) delivery offers distinct advantages, including higher editing precision, lower off-target effects, and elimination of genomic integration risks [2–4]. Consequently, the RNP-based CRISPR/Cas9 system has been widely adopted across a variety of organisms, enabling applications including resistance marker-free knockouts in fungi, high-efficiency precise editing in plant protoplasts, and the generation of stable mutant animal models such as zebrafish for functional analysis in vivo [5–7]. However, the production of high-purity Cas9 protein remains challenging due to its large size (158 kDa) and the complexities of conventional purification workflows [8].
Conventional protocols often involve multiple purification steps, such as affinity, ion-exchange, and size-exclusion chromatography, as well as protease cleavage to remove fusion tags [9,10]. These multi-step processes not only increase time and cost but also lead to significant protein loss [11]. To overcome these limitations, we developed a single-step purification method using a ubiquitin fusion strategy. The Ub tag significantly enhances soluble expression of the fusion protein in E. coli [12]. Furthermore, it can be cleaved by endogenous deubiquitylases in eukaryotic cells [13], eliminating the need for in vitro tag removal and thereby avoiding operational complexity and low recovery yields. The Ub fusion approach simplifies the purification while maintaining Cas9’s structural and functional integrity. Comparative analysis confirms that the purified Ub-Cas9 exhibits nuclease activity comparable to that of commercial Cas9 in vivo.
This protocol describes the construction of His-Ub-Cas9 expression plasmid, its expression in E. coli BL21(DE3), and one-step purification using Ni-NTA affinity chromatography. We also provide detailed procedures for in vivo functional validation in zebrafish embryos. The entire process can be completed within one week and yields over 8 mg/L of Cas9 protein with >95% purity and robust in vivo editing activity.
In summary, we present a streamlined, efficient, and cost-effective method for obtaining high-purity functional Cas9 protein, which will facilitate its broader application in genome editing and gene therapy research.
Materials and reagents
Biological materials
1. pET-28b-Cas9-His plasmid (Addgene, catalog number: 47327)
2. pHUE plasmid [12]
3. pCMV-Tag2B (Agilent Technologies, catalog number: 211172)
4. E. coli BL21 (DE3) cells (NEB, catalog number: C2527H)
5. E. coli DH5α cells (NEB, catalog number: C2988J)
Reagents
1. Phanta Max Super-Fidelity DNA Polymerase (Vazyme, catalog number: P505)
2. TIANgel Purification kit (TIANGEN, catalog number: DP219-03)
3. Restriction enzyme BamH I, 10× K buffer (TAKARA, catalog number: 1010S)
4. Restriction enzyme Hind III (TAKARA, catalog number: 1060S)
5. Exnase II, 5× CE II buffer (Vazyme, catalog number: C112)
6. 2× Hieff® PCR Master Mix (with dye) (Yeasen, catalog number: 10101)
7. TIANprep Mini Plasmid kit (TIANGEN, catalog number: DP103-03)
8. Tryptone (Beyotime, catalog number: ST799)
9. Yeast extract (Beyotime, catalog number: ST969)
10. Sodium chloride (NaCl) (MCKLIN, catalog number: S805275)
11. Agar powder (Solarbio, catalog number: A8190)
12. Ampicillin (Beyotime, catalog number: ST008)
13. 50× TAE buffer (Beyotime, catalog number: ST716)
14. Agarose (Yeasen, catalog number: 10208ES60)
15. Super Red DNA gel stain (Biosharp, catalog number: BS354B)
16. 10× DNA loading buffer (TAKARA, catalog number: SD0012)
17. DL 2000 DNA marker (TAKARA, catalog number: 3427A)
18. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Yeasen, catalog number: 10902ES08)
19. M5 protease inhibitor cocktail, EDTA-free (100× DMSO) (Mei5bio, catalog number: MF182-plus-0)
20. Triton X-100 (Biosharp, catalog number: BS084)
21. Glycerol (MACKLIN, catalog number: G810575)
22. Imidazole (Sigma-Aldrich, catalog number: I2399)
23. 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) (Sigma, catalog number: H3375)
24. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (MACKLIN, catalog number: T917415)
25. Potassium hydroxide (KOH) (XILONG, catalog number: 10202101)
26. Potassium chloride (KCl) (XILONG, catalog number: 10200501)
27. Coomassie Blue R-250 (AMRESCO, catalog number: C0472)
28. Methanol (XILONG, catalog number: 12801001)
29. Acetic acid (XILONG, catalog number: 12705701)
30. Coomassie Blue G-250 (Beyotime, catalog number: ST030)
31. Ethanol (XILONG, catalog number: 12803401)
32. Phosphoric acid (XILONG, catalog number: 12704101)
33. Glycine (Solarbio, catalog number: G8200)
34. Tris base (Solarbio, catalog number: T80)
35. Sodium dodecyl sulfate (SDS) (Solarbio, catalog number: S8010)
36. 1 M Tris-HCl (Solarbio, catalog number: T1020)
37. 1.5 M Tris-HCl (Solarbio, catalog number: T1010)
38. 30% acrylamide-bisacrylamide (Biosharp, catalog number: BL513b)
39. Ammonium persulfate (APS) (MACKLIN, catalog number: 801037)
40. N,N,N’,N’-Tetramethylethylenediamine (TEMED) (Solarbio, catalog number: T8090)
41. Isopropyl alcohol (XILONG, catalog number: 12802501)
42. SDS loading buffer (5×) (Beyotime, catalog number: P0015)
43. M5 HiClear prestained protein ladder (10–180 kDa) (Mei5bio, catalog number: MF212)
44. Sodium phosphate dibasic (Na2HPO4) (XILONG, catalog number: 10105901)
45. Disodium phosphate dodecahydrate (Na2HPO4·12H2O) (XILONG, catalog number: 1010590104204)
46. Potassium dihydrogen phosphate (KH2PO4) (XILONG, catalog number: 10204401)
47. Calcium chloride (CaCl2) (GHTECH, catalog number: 111-2003)
48. Sodium bicarbonate (NaHCO3) (XILONG, catalog number: 10104201)
49. Cas9-N-NLS nuclease (Genscript, catalog number: Z03388)
50. Sodium hydroxide (NaOH) (XILONG, catalog number: 10103101)
51. T7 Endonuclease I (Vazyme, catalog number: EN303)
52. Liquid nitrogen (local distributor)
Solutions
1. Luria-Bertani (LB) medium (see Recipes)
2. 100 mg/mL ampicillin (see Recipes)
3. LB medium with ampicillin (see Recipes)
4. LB agar plate with ampicillin (see Recipes)
5. 1 M IPTG (see Recipes)
6. Lysis buffer (see Recipes)
7. Elution buffer (see Recipes)
8. Storage buffer (see Recipes)
9. 10% APS (see Recipes)
10. 5× Tris-glycine buffer (see Recipes)
11. 10 mg/mL BSA (see Recipes)
12. Coomassie blue staining solution (see Recipes)
13. Destaining solution (see Recipes)
14. 1× PBS (see Recipes)
15. 10× PBS (see Recipes)
16. 5× Bradford reagent (see Recipes)
17. Embryo medium (see Recipes)
18. 50 mM NaOH (see Recipes)
19. 100 mM Tris-HCl (pH 8.0) (see Recipes)
Recipes
1. Luria-Bertani (LB) medium
| Reagent | Final concentration | Amount |
|---|---|---|
| Tryptone | 1% (w/v) | 10 g |
| Yeast extract | 0.5% (w/v) | 5 g |
| NaCl | 1% (w/v) | 10 g |
| ddH2O | n/a | ~980 mL |
| Total | n/a | 1 L |
Autoclave at 121 °C for 20 min. Store at room temperature.
2. 100 mg/mL ampicillin
| Reagent | Final concentration | Amount |
|---|---|---|
| Ampicillin | 100 mg/mL | 1 g |
| ddH2O | n/a | 10 mL |
Sterilize the solution with a 0.22 μm syringe filter. Store at -20 °C.
3. LB medium with ampicillin
| Reagent | Final concentration | Amount |
|---|---|---|
| LB medium | 1× | 50 mL |
| 100 mg/mL ampicillin | 100 μg/mL | 50 μL |
Store at 4 °C.
4. LB agar plate with ampicillin
| Reagent | Final concentration | Amount |
|---|---|---|
| LB broth | 25 g/L | 25 g |
| Agar | 15 g/L | 15 g |
| 100 mg/mL ampicillin | 100 μg/mL | 1 mL |
| ddH2O | n/a | ~980 mL |
| Total | n/a | 1 L |
a. Dissolve LB broth in ~980 mL of ddH2O. Transfer the medium to a 1-L glass bottle.
b. Add 15 g of agar, drop in a stir bar, and autoclave at 121 °C for 20 min.
c. Stir the medium and allow to cool to 50–60 °C.
d. Add 1 mL of 100 mg/mL ampicillin solution. Stir well.
e. Transfer 20 mL of the solution to 10-cm Petri dishes using a serological pipette.
f. Allow the agar plates to cool and dry in a laminar flow hood for 30 min.
g. Store the plates at 4 °C in a plastic bag.
5. 1 M IPTG
| Reagent | Final concentration | Amount |
|---|---|---|
| IPTG | 1 M | 2.4 g |
| ddH2O | n/a | 10 mL |
a. Weigh 2.4 g of IPTG powder and place it into a 15 mL tube.
b. Add 8 mL of ddH2O. Mix thoroughly until dissolved, then bring the volume up to 10 mL with ddH2O.
c. Sterilize the solution by filtration through a 0.22 μm syringe filter.
d. Aliquot into small volumes (1 mL per aliquot) and store at -20 °C.
6. Lysis buffer
| Reagent | Final concentration | Amount |
|---|---|---|
| 1 M Tris-HCl, pH 7.5 | 20 mM | 4 mL |
| NaCl | 500 mM | 5.844 g |
| Glycerol | 5% (v/v) | 10 mL |
| Imidazole | 40 mM | 0.544 g |
| ddH2O | n/a | ~170 mL |
| Total | n/a | 200 mL |
a. Weigh 5.844 g of NaCl and 0.544 g of imidazole and transfer to a clean 250 mL beaker.
b. Add about 170 mL of ddH2O and stir using a magnetic stir bar until completely dissolved.
c. Add 4 mL of 1 M Tris-HCl (pH 7.5) and 10 mL of glycerol to the solution and mix completely.
d. Adjust the pH to 7.5 with 1 M HCl or 1 M NaOH, then bring the final volume to 200 mL with ddH2O.
e. Filter the buffer through a 0.22 μm syringe filter and store at 4 °C.
7. Elution buffer
Elution buffer 1: Lysis buffer with 80 mM imidazole
Elution buffer 2: Lysis buffer with 500 mM imidazole
Filter the buffer through a 0.22 μm syringe filter. Store at 4 °C.
8. Storage buffer
| Reagent | Final concentration | Amount |
|---|---|---|
| HEPES | 20 mM | 0.952 g |
| KCl | 150 mM | 2.235 g |
| Glycerol | 10% (v/v) | 20 mL |
| 500 mM TCEP | 1 mM | 400 μL |
| ddH2O | n/a | ~160 mL |
| Total | n/a | 200 mL |
a. Dissolve 0.143 g of TCEP in 1 mL of ddH2O to prepare a 500 mM stock solution. Store at 4 °C for several weeks.
b. Prepare the buffer by combining the components above.
c. Include 400 μL of the 500 mM TCEP stock to a final concentration of 1 mM.
d. Adjust the pH to 7.5 with 1 M KOH.
e. Filter the buffer through a 0.22 μm syringe filter. Store at 4 °C for up to one week.
9. 10% APS
| Reagent | Final concentration | Amount |
|---|---|---|
| APS | 10% (w/v) | 1 g |
| ddH2O | n/a | 10 mL |
Store at 4 °C for up to one week.
10. 5× Tris-glycine buffer
| Reagent | Final concentration | Amount |
|---|---|---|
| Tris | 0.125 M | 15.1 g |
| Glycine | 1.25 M | 94 g |
| SDS | 0.5% (w/v) | 5.0 g |
| ddH2O | n/a | ~980 mL |
| Total | n/a | 1 L |
11. 10 mg/mL BSA
| Reagent | Final concentration | Amount |
|---|---|---|
| BSA | 10 mg/mL | 0.1 g |
| ddH2O | n/a | 10 mL |
12. Coomassie blue staining solution
| Reagent | Final concentration | Amount |
|---|---|---|
| Coomassie blue R-250 | 0.05% (w/v) | 0.5 g |
| Methanol | 50% (v/v) | 500 mL |
| Acetic acid | 10% (v/v) | 100 mL |
| ddH2O | n/a | ~380 mL |
| Total | n/a | 1 L |
Filter through filter paper after preparation and store at room temperature.
13. Destaining solution
| Reagent | Final concentration | Amount |
|---|---|---|
| Methanol | 50% (v/v) | 500 mL |
| Acetic acid | 10% (v/v) | 100 mL |
| ddH2O | n/a | ~380 mL |
| Total | n/a | 1 L |
14. 1× PBS
| Reagent | Final concentration | Amount |
|---|---|---|
| NaCl | 137 mM | 8 g |
| KCl | 2.7 mM | 0.2 g |
| Na2HPO4 | 10 mM | 1.42 g |
| KH2PO4 | 2 mM | 0.27 g |
| ddH2O | n/a | ~980 mL |
| Total | n/a | 1 L |
a. Adjust the pH to 7.4 by adding HCl, then bring the final volume to 1 L with ddH2O.
b. Autoclave at 121 °C for 20 min and store at room temperature.
15. 10× PBS
| Reagent | Final concentration | Amount |
|---|---|---|
| NaCl | 1.37 M | 80 g |
| KCl | 27 mM | 2 g |
| Na2HPO4·12H2O | 80 mM | 29 g |
| KH2PO4 | 20 mM | 2.7 g |
| ddH2O | n/a | ~980 mL |
| Total | n/a | 1 L |
Adjust the pH to 7.4 by adding HCl, then bring the final volume to 1 L with ddH2O.
16. 5× Bradford reagent
| Reagent | Final concentration | Amount |
|---|---|---|
| Coomassie blue G-250 | 0.05% (w/v) | 50 mg |
| 95% ethanol | 23.75% (v/v) | 25 mL |
| 85% H3PO4 | 42.5% (v/v) | 50 mL |
| ddH2O | n/a | ~20 mL |
| Total | n/a | 100 mL |
a. Dissolve 50 mg of Coomassie Brilliant Blue G-250 in 25 mL of 95% ethanol with stirring.
b. Add 50 mL of 85% H3PO4.
c. Bring the volume to 100 mL with ddH2O.
d. Filter through filter paper and store at 4 °C.
17. Embryo medium
| Reagent | Final concentration | Amount |
|---|---|---|
| NaCl | 60 mM | 3.5 g |
| KCl | 0.67 mM | 0.05 g |
| NaHCO3 | 2.4 mM | 0.2 g |
| CaCl2 | 0.9 mM | 0.1 g |
| ddH2O | n/a | ~980 mL |
| Total | n/a | 1 L |
Store at room temperature.
18. 50 mM NaOH
| Reagent | Final concentration | Amount |
|---|---|---|
| NaOH | 50 mM | 0.1 g |
| ddH2O | n/a | 50 mL |
19. 100 mM Tris-HCl (pH 8.0)
| Reagent | Final concentration | Amount |
|---|---|---|
| Tris base | 100 mM | 0.606 g |
| ddH2O | n/a | 50 mL |
a. Dissolve Tris base in 40 mL of ddH2O.
b. Stir the solution and adjust the pH to 8.0 with HCl.
c. Adjust the final volume to 50 mL with ddH2O.
Laboratory supplies
1. Pipette tips: 20, 200, 1,000 μL (Biosharp, catalog numbers: T-001-10, T-001-200, T-001-1000)
2. Pipette (Eppendorf, catalog numbers: 4924000029, 4924000053, 4924000088)
3. 200 μL PCR tube (LABSELECT, catalog number: PST-0208-FT-C)
4. 1.5 and 2 mL microcentrifuge tube (Biofil, catalog numbers: CFT000015, CFT000020)
5. 15 and 50 mL conical tube (Biosharp, catalog numbers: CT-002-15, CT-002-50)
6. 250 mL and 1 L Erlenmeyer flask (Biosharp, catalog numbers: 57202, 57402)
7. 250 mL and 1 L beaker (Biosharp, catalog numbers: BS-LFB-250, BS-LFB-1000)
8. 0.22 μm syringe filter (Millipore, catalog number: SLGVR33RS)
9. 20 mL syringe (Beyotime, catalog number: FS820-40pcs)
10. 10 cm Petri dish (Yeasen, catalog number: 84009ES50)
11. Disposable plastic cuvette (Yeasen, catalog number: 84201ES20)
12. Ni-NTA resin (GE Healthcare, catalog number: 17-5318-02)
13. PD-10 desalting column (GE Healthcare, catalog number: 17-0851-01)
14. 30 kDa MWCO (Amicon Ultra, catalog number: UFC901024)
15. Glass capillary tube (WPI, catalog number: 1B100F-4)
16. Microloader pipette tip (Eppendorf, catalog number: 5242 956.003)
17. Surgical scalpel (Beyotime, catalog number: FS205)
18. PARAFILM® M (Merck, catalog number: P7793)
Equipment
1. Thermal cycler (Eppendorf, catalog number: 6337000078)
2. Water bath (Tongjun, model: HH-11-2)
3. Centrifuge (Eppendorf, model: 5418R/5804R)
4. Incubator (YETUO, model: XMA-600)
5. Shaker incubator (NOKI, model: TS-200B)
6. 28 °C incubator (LICHEN, model: LC-HN-25S)
7. Analytical balance (Sartorius, model: GL124-1SCN)
8. UV spectrophotometer (SHIMADZU, model: UV1900i)
9. SevenEasy pH meter (METTLER TOLEDO, model: S20K)
10. Diaphragm vacuum pump (Xiangmingjie, model: XMJ-15T)
11. Nanodrop one (Thermo Fisher, catalog number: 840-317400)
12. DNA electrophoresis system (JUNYI, model: JT300C)
13. Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad, catalog number: 165-8000)
14. Bio-Rad PowerPac HV power supply (Bio-Rad, catalog number: 164-5050)
15. DNA agarose gel imaging equipment (Tanon, model: 2500R)
16. Ultrasonic cell breaker (Yetuo, model: YT-JY96-IIN)
17. Flaming/brown micropipette puller (SUTTER, model: P-97)
18. Microinjector (Eppendorf, catalog number: 5176)
19. Stereomicroscope (CNOPTEC, model: SZ810)
20. Magnetic hotplate stirrer (SCILOGEX, catalog number: 80301321159999)
21. Vortex mixer (YEASEN, catalog number: 80172ES01)
22. Milli-Q® EQ 7000 water purification system (Millipore, catalog number: ZEQ7000T0C)
23. Autoclave (Boxun, model: YXQ-100SII)
24. Fume hood (Esco, model: Frontier Acela)
25. Drying oven (Jinghongsh, model: DHG-9070A)
26. Metal bath (Yooning, model: GH-100)
Software and datasets
1. SnapGene, for molecular cloning design and DNA sequence analysis (version 6.0.2)
2. GraphPad Prism 9, for statistics and data visualization (version 9.3.1)
3. ImageJ, for image processing (version 1.53a)
Procedure
文章信息
稿件历史记录
提交日期: Nov 19, 2025
接收日期: Dec 29, 2025
在线发布日期: Jan 13, 2026
出版日期: Feb 5, 2026
版权信息
© 2026 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
如何引用
Duan, X., Zhou, Z. and Mao, A. (2026). A One-Step Method for Efficient Purification of Functional Cas9 Protein. Bio-protocol 16(3): e5594. DOI: 10.21769/BioProtoc.5594.
Download Citation in RIS Format
分类
生物化学 > 蛋白质 > 分离和纯化
发育生物学 > 基因组编辑 > 靶向整合
生物科学 > 生物技术 > CRISPR/Cas9
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