1. Because the BL21 bacterial strain contains the gene for T7 RNA polymerase, and
can therefore produce it. According to the plasmid map, the Cas9 gene expression
relies on activation of the T7 promoter. Other bacterial strains without T7 RNA
polymerase cannot activate the expression of the desired Cas9.
2. Low-concentration imidazole does not form enough competition with the Cas9 Histag in binding with the Ni-NTA resin but instead washes oI non-Cas9 proteins from
the system. After several steps of elution, other proteins are mostly removed,
leaving mostly pure Cas9 on the beads. At this stage, with a high-concentration
wash, the imidazole can displace the Cas9 from the beads, and the Cas9 can be
collected. A single high-concentration imidazole wash would elute both non-Cas9
and Cas9 proteins at the same time, and Cas9 would be low in purity.
3. The purified Cas9 protein can be delivered into cells and work with sgRNA to enable
gene editing. For example, sgRNA complementary to the mutant HBB sequence
could guide Cas9 to cut the faulty section and the cell would restore itself to the
non-mutant HBB sequence, providing a treatment for sickle cell anemia.
4. Cas9 protein was extracted from E. coli cells and purified by immobilized metal
aIinity chromatography (IMAC). The cell pellet collected from 50 mL bacterial
culture was resuspended in 10 mL of BuIer A (20 mM HEPES, pH 7.5, 300 mM NaCl)
with 0.1 mM PMSF (protease inhibitor) in a 50 mL Falcon tube by vortexing until no
cell clumps remained. The resuspended cells were lysed at 750 bar for 2 min in a
high-pressure cell crusher pre-cooled to 5 °C. The lysate was then clarified by
centrifugation at 11,000 × g for 30 min; the supernatant was filtered through a 0.45
µm filter. A PD-10 column with filter attached was clamped to a stand. All
subsequent liquids were added slowly. The filter was rinsed with 5 mL 20% ethanol
once and 10 mL double-distilled water twice. 2 mL of Ni-NTA resin slurry (gently
shaken homogeneous) was transferred to the PD-10 column. The 20% ethanol that
preserved the resin was drained and the resin was thoroughly rinsed twice, each
with 5 mL of double-distilled water, then equilibrated with 2 mL of BuIer A.
Approximately 1 mL of BuIer A was added to keep the resin hydrated. 5 mL of
clarified cell lysate was added to the PD-10 column and incubated at room
temperature on a shaker at low speed for 10 min. After incubation, the PD-10
column was re-clamped to the stand. 1 mL of flowthrough (labeled FT) was
collected in a 1.5 mL EP tube and the rest of the liquid was drained into a liquid
waste bin. The column was washed with 1 mL of BuIer A. A five-step gradient of
BuIer B (20 mM HEPES, pH 7.5, 300 mM NaCl, 500 mM imidazole; 5, 15, 25, 50, and
100%) diluted with BuIer A was prepared in a volume of 1 mL each (5%: 950 µL
BuIer A + 50 µL BuIer B; 15%: 850 µL BuIer A + 150 µL BuIer B; 25%: 750 µL BuIer
A + 250 µL BuIer B; 50%: 500 µL BuIer A + 500 µL BuIer B; 100%: 1000 µL BuIer B);
the gradient was used to sequentially elute the protein with increasing
concentrations of BuIer B. The five eluted fractions were collected and labeled E1,
E2, E3, E4 and E5 respectively. The Ni-NTA resin was then washed with 1 mL BuIer
C (20 mM HEPES, pH 7.5, 300 mM NaCl, 1 M imidazole), 5 mL BuIer A, 5 mL doubledistilled water, and 5 mL 20% ethanol respectively (all fractions from the rinsing
process were discarded). The resin was resuspended in 2 mL of 20% ethanol for
future use.