Expression of azido-Cas9 mutants

E. coli BL21 cells transformed with pET28a-Cas9-Mutants and pUltra- MjPolyRS were cultivated in 2YT medium. Resuspend a single colony in liquid culture with antibiotic to produce a starter culture
Inoculate starter culture at a 1:100 dilution into expression media containing antibiotic (Amp+Spec). Incubate at 37°C with shaking until OD600 reaches 0.3.
AeF was added at the final concentration of 1mM. The cells were continuing growth until the OD600 reached 0.6~0.8 and then 0.2 mM IPTG (Sigma) was added.
The culture temperature was lowered to 18°C for overnight culture.

Purification of azido-Cas9 mutants

1. Ni-NTA purification
Solution prepared for Ni-NTA purification

Lysis buffer: 20mM Tris-HCl 1M KCl PH8.0

Wash buffer: 20mM Tris-HCl 1M KCl 20mM imidazole PH8.0

Elution buffer: 20mM Tris-HCl 1M KCl 250mM imidazole PH8.0

1) Cells were grown overnight and harvested by centrifugation at 6,000 rpm at 4°C.

2) Cell were resuspended in lysis buffer containing 1x PMSF to prevent protein degradation and sonicate on ice.

3) Centrifuge lysate at 18,000 x g for 20-30 minutes at 4°C to pellet the cellular debris.

4) Balance the Ni-NTA resin with lysis buffer and load the supernatant at step 3 on the column to make it flow through with gravity. Repeat loading step for 3 times for making sure the protein could bind the Ni matrix tightly.

5) Wash the resin with 5-10 times column volume with wash buffer.

6) Elution with elution buffer (Typically，5-10 ml elution buffer was used for 500ml expression volume)

7) Concentration and buffer exchanged to buffer A（20mM Tris-HCl 300mM KCl PH8.0） with 50kd Millipore Amicon Ultra-50 for further purification.

8) Purity and concentration of Cas9 protein were analyzed by SDS- polyacrylamide gel electrophoresis.

2. Cas9 purification by Ion exchange chromatography

Solution and equipment prepared for IEC purification

Buffer A: 20mM Tris-HCl 300mM KCl PH8.0

Buffer B: 20mM Tris-HCl 1M KCl PH8.0

1) HitrapTM SP HP, (GE Healthcare 5ml) was used on AKTA pure for purification.

2) Balanced the column with buffer A at 2ml/min.

3) Wash the injection loop and inject the concentrated sample into the injection loop.

4) After the monitor of UV visible and condensation smooth, set the elution buffer with gradient concentration buffer B (100% B in 10-20min) and collect the tube according to the A280/A260

5) Concentration and buffer exchanged to PBS with 50kd Millipore Amicon Ultra-50 for further purification.

6) Purity and concentration of Cas9 protein were analyzed by SDS- polyacrylamide gel electrophoresis.

Figure 1 Cas9 Mutant purification by IEC (1L and 200ml expression volume)

3. Cas9 purification by Size Exclusion chromatography

Solution and equipment prepared for SEC purification

PBS

Superdex 200, GE Healthcare

1) Balanced the column with PBS at 0.5ml/min.

2) Wash the injection loop and inject the concentrated sample into the injection loop.

3) Continue wash the column with PBS and collect the tube according to the A280/A260.

4) Purity and concentration of Cas9 protein were analyzed by SDS- polyacrylamide gel electrophoresis.

Figure 2 Cas9 Mutant purification by SEC (1L and 200ml expression volume)

Figure 3 Cas9 Mutant purity was analyzed by SDS-PAGE