A Robust Expression and Purification Method for Production of SpCas9-GFP-MBP Fusion Protein for In Vitro Applications

Genome editing using the CRISPR/Cas9 system is one of the trendiest methodologies in the scientific community. Many genome editing approaches require recombinant Streptococcus pyogenes Cas9 (SpCas9) at some point during their application, for instance, for in vitro validation of single guide RNAs (SgRNAs) or for the DNA-free editing of genes of interest. Hereby, we provide a simple and detailed expression and purification protocol for SpCas9 as a protein fused to GFP and MBP. This protocol improves protein yield and simplifies the purification process by overcoming the frequently occurring obstacles such as plasmid loss, inconsistent protein expression levels, or inadequate protein binding to affinity resins. On average, this protocol yields 10 to 30 mg of purified, active, His6−MBP−SpCas9 NLS−GFP protein. The purity addressed through SDS-PAGE is > 80%.


Introduction
The CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein 9) system is the most efficient, flexible, and broadly used technology for genome editing (GE). CRISPR/Cas9 based systems involve a powerful and continuously growing number of tools for a variety of applications, such as targeted DNA mutagenesis, transcriptional control of gene expression, nucleic acid dynamics imaging and epigenetic manipulation [1,2], as well as diagnostic assays [3].
While DNA-free methodologies have become popular for GE of animal cells [4,5], in plants, GE is frequently implemented using transgenesis to introduce a gene construct that allows the expression of Cas9 and the single guide RNAs (sgRNA). Stable transformed plants harboring the intended mutation can be subsequently self-pollinated or crossed to segregate the incorporated DNA construct to obtain transgene-free plants [6,7]. However, DNA-free strategies can be used in plants to guarantee the non-incorporation of foreign DNA into the genome. Instead of using DNA constructs, Cas9 can be directly delivered into the cells in the form of a protein or messenger RNA (mRNA), together with the in vitro synthesized sgRNAs [8][9][10][11][12][13][14].
Regardless of the method for delivering the editing machinery into the cells, most in vivo applications of CRISPR-based technologies require prior in vitro testing of sgRNA efficiency on target recognition using Cas9-sgRNA ribonucleoprotein (RNP). This is particularly important when working with plants, where editing performance can be highly affected by the transformation and tissue culture efficiencies of the plant species [6,7]. Even though Cas9 can be commercially acquired, the cost of this reagent can still be too high for emerging research groups or researchers working in low-income countries.
Although a number of protocols for Streptococcus pyogenes Cas9 expression and purification have been published (Table 1), we provide an improved step by step protocol that Table 1. Cas9 expression and purification protocols.
To our knowledge, this is the first report of a detailed Cas9 purification protocol that includes troubleshooting for each stage of the procedure. Moreover, alternative procedures are included in case not all of the listed equipment is available in the lab (Section 5).

Experimental Design
The protein was expressed from plasmid pMJ922 [18] obtained from Addgene ( Figure 1A). This plasmid allows the expression of SpCas9 fused in-frame with a C-terminal hemagglutinin (HA) epitope tag, a bipartite nuclear localization signal (NLS) sequence, a green fluorescent protein (GFP) polypeptide, and an additional monopartite NLS at the very C-terminus (SpCas9 NLS−GFP). SpCas9 NLS−GFP is expressed as a fusion protein with an N-terminal hexahistidine-maltose binding protein (His6−MBP) affinity tag ( Figure 1) that can be removed by cleavage with Tobacco etch virus protease (TEV-P) during purification, depending on the desired application. The time needed to complete each stage is summarized in Table 2.
hemagglutinin (HA) epitope tag, a bipartite nuclear localization signal (NLS) sequence, a green fluorescent protein (GFP) polypeptide, and an additional monopartite NLS at the very C-terminus (SpCas9 NLS−GFP). SpCas9 NLS−GFP is expressed as a fusion protein with an N-terminal hexahistidine-maltose binding protein (His6−MBP) affinity tag (Figure 1) that can be removed by cleavage with Tobacco etch virus protease (TEV-P) during purification, depending on the desired application. The time needed to complete each stage is summarized in Table 2.   3.2.1. Bacterial Lysis 1. Resuspend 3-6 g of cell pellet in IMAC buffer A (50 mM TRIS pH 7.5, 500 mM NaCl, 5 mM MgCl2) in a 5:1 buffer:pellet ratio. Perform an initial lysis step by three cycles of freezing/thawing the pellet at −80 °C. Transfer the suspension to a small beaker and add lysozyme to a final concentration of 0.1 mg/mL, 1 mM DTT final concentration, additional MgCl2, 1:500 v/v from 1 M stock solution, a protease inhibitor tablet (Roche, Cat. no. 05892970001) and 1 μL of benzonase (25 kU/μL, Pierce, Cat. 88701).
CRITICAL STEP: using benzonase instead of DNase greatly simplifies further purification steps. Benzonase digests all forms of nucleic acids, which can affect purity during IMAC and binding to the ion exchange resin if left undigested.
Contamination with nucleic acids from E. coli can be problematic for Cas9 purification because these molecules can interfere with the binding capacity of Cas9 to the IEX resin due to PI alteration. This problem can be addressed by using RNase in addition to DNase or, alternatively, benzonase, which exhibits both DNase and RNase activity.  CRITICAL STEP: the pellet should be greenish due to the GFP expression ( Figure 2B).

7.
Prepare two plates for this purpose. 3. Using a sterile spatula, scrape all the bacteria from a plate and resuspend the cells in 5 mL LB media. Add this suspension to a 2 L Erlenmeyer containing 500 mL TB media (24 g/L yeast extract, 20 g/L tryptone, 4 mL/L glycerol, 17 mM KH2PO4, 72 mM K2HPO4) supplemented with 200 μg/mL ampicillin and 34 μg/mL chloramphenicol. Incubate at 37 °C and 200 rpm until absorbance at 600 nm reach 0. CRITICAL STEP: the pellet should be greenish due to the GFP expression ( Figure  2B). 7.
PAUSE STEP: pellets can be stored at −80 °C for several months until use.

Protein Purification
PAUSE STEP: pellets can be stored at −80 • C for several months until use.
Prepare two plates for this purpose. 3. Using a sterile spatula, scrape all the bacteria from a plate and resuspend the cells in 5 mL LB media. Add this suspension to a 2 L Erlenmeyer containing 500 mL TB media (24 g/L yeast extract, 20 g/L tryptone, 4 mL/L glycerol, 17 mM KH2PO4, 72 mM K2HPO4) supplemented with 200 μg/mL ampicillin and 34 μg/mL chloramphenicol. Incubate at 37 °C and 200 rpm until absorbance at 600 nm reach 0. CRITICAL STEP: the pellet should be greenish due to the GFP expression ( Figure  2B). 7.
PAUSE STEP: pellets can be stored at −80 °C for several months until use.  Contamination with nucleic acids from E. coli can be problematic for Cas9 purification because these molecules can interfere with the binding capacity of Cas9 to the IEX resin due to PI alteration. This problem can be addressed by using RNase in addition to DNase or, alternatively, benzonase, which exhibits both DNase and RNase activity. Contamination with nucleic acids from E. coli can be problematic for Cas9 purification because these molecules can interfere with the binding capacity of Cas9 to the IEX resin due to PI alteration. This problem can be addressed by using RNase in addition to DNase or, alternatively, benzonase, which exhibits both DNase and RNase activity. If free SpCas9−GFP is needed, buffer exchange with a desalting column can be substituted by dialysis against IEX buffer A in the presence of TEV-P. Collect the protein eluted from IMAC (Section 3.2.2) and add DTT to 1 mM final concentration, EDTA to 1 mM final concentration, and 1 mg of TEV-P for every mg of protein.

Protein Purification
Place the protein solution in dialysis tubing with a cutoff >12 kDa. Perform dialysis overnight at 4 • C against buffer IEX A. Place the dialysis tubing inside a 500 mL graduated cylinder with constant stirring for this purpose.
To separate free SpCas9−GFP from His−MBP, perform a second IMAC (IMAC2) using the same conditions as the first IMAC (Section 3.2.2). His−MBP and SpCas9−GFP bound to MBP will be retained in the IMAC column, while free SpCas9−GFP will be present in the flow through ( Figure S2).
Buffer exchange SpCas9−GFP obtained from this step as described in Section 3.2.3 and proceed with Section 3.2.4.
If free SpCas9−GFP is needed, buffer exchange with a desalting column can be substituted by dialysis against IEX buffer A in the presence of TEV-P. Collect the protein eluted from IMAC (Section 3.2.2) and add DTT to 1 mM final concentration, EDTA to 1 mM final concentration, and 1 mg of TEV-P for every mg of protein.
Place the protein solution in dialysis tubing with a cutoff > 12 kDa. Perform dialysis overnight at 4 °C against buffer IEX A. Place the dialysis tubing inside a 500 mL graduated cylinder with constant stirring for this purpose.
To separate free SpCas9−GFP from His−MBP, perform a second IMAC (IMAC2) using the same conditions as the first IMAC (Section 3.2.2). His−MBP and SpCas9−GFP bound to MBP will be retained in the IMAC column, while free SpCas9−GFP will be present in the flow through ( Figure S2).
Buffer exchange SpCas9−GFP obtained from this step as described in Section 3.2.3 and proceed with Section 3.2.4.  14. Store the protein at a final concentration > 1 mg/mL (usually the protein concentration is higher than 1 mg/mL after buffer exchange). Prepare single use aliquots and flash freeze in liquid nitrogen. Store at −80 • C. The protein remains active for over a year.

2.
Allow the reaction to equilibrate for 15 min at room temperature. Add 300 ng of the PCR product, briefly vortex and spin. Incubate at 37 • C for 2 h. 3.
Add 1 µL of 20 mg/mL proteinase K and incubate for 10 min at 37 • C. Analyze in agarose gel. The agarose percentage should be according to the expected DNA fragment sizes after cleavage. An example of a typical activity assay is shown in Figure 4. 3. Add 1 μL of 20 mg/mL proteinase K and incubate for 10 min at 37 °C. Analyze in agarose gel. The agarose percentage should be according to the expected DNA fragment sizes after cleavage. An example of a typical activity assay is shown in Figure 4.

Protocol Alternatives and Troubleshooting
Protocol alternatives can be found in Table 4 and troubleshooting in Table 5. A. Perform IMAC with the column connected to a peristaltic pump. Follow absorbance at 280 nm using a spectrophotometer. Equilibrate the IMAC column with 10 CV on IMAC buffer A and measure the buffer absorbance before loading your protein sample. Make sure that this value is reached again after extensively washing bound protein. B. Perform batch incubation with IMAC resin. Follow the absorbance at 280 nm using a spectrophotometer. Equilibrate the resin in IMAC buffer A and measure the buffer absorbance before loading your protein sample. Make sure that this value is reached again after extensively washing bound protein. Step Suggested Procedure Alternative
A. Buffer exchange the protein using centrifugal filter units with a cutoff >50 kDa and perform several dilution/concentration steps. Use IEX buffer A as the diluting solution.

IEX
Cation exchange SpHp HighTrap column (GE, Cat. GE29-0513-24) connected to ÄKTA FPLC system (GE, Cat. no.: 29-0598-78 AB) A. Perform IEX with the column connected to a peristaltic pump. Follow the absorbance at 280 nm using a spectrophotometer. Equilibrate the IMAC column with 10 CV on IEX buffer A and measure the buffer absorbance before loading your protein sample. Make sure that this value is reached again after extensively washing bound protein. Perform elution in a single step with 1 M NaCl concentration or in several steps with increasing NaCl concentrations.
A. Buffer exchange the protein using centrifugal filter units with a cutoff >50 kDa and perform several dilution/concentration steps. Use storage buffer as the diluting solution. B. Use a Superdex 200 16/600 size exclusion column (GE Healthcare), equilibrated in storage buffer for the final polishing step.

Problem Possible Explanation Solution
No protein expression Plasmid loss Retransform BL21 Rosetta or use another aliquot from your bacterial stock. Check plasmid presence in your bacterial stock.

Incomplete wash of nonspecific proteins
Do not elute protein until buffer absorbance from the washing fraction reaches the starting value. Try performing a washing step with IMAC buffer A containing 5 mM imidazole. This can lead to some Cas9 washing as well.

No protein binding to IEX column
Nucleic acid contamination. Treat the protein fraction with benzonase.