Streptococcus pyogenes

Versions of Cas9 derived from three different species have been exploited to edit genes in human cells. These Cas9 proteins recognize different PAM sequences. Cas9 originated from Streptococcus pyogenes (SpCas9) recognizes 5′-NGG-3′ PAM sequences and, to a lesser extent, 5′-NAG-3′. Cas9 from Streptococcus thermophilus (StCas9) (Cong et al., 2013 (link)) and that from Neisseria meningitidis (NmCas9) (Hou et al., 2013 (link)) recognizes 5′-NNAGAAW-3′ (W = A or T) and 5′-NNNNGMTT-3′ (M = A or C), respectively. The degeneracy in PAM recognition by Cas9 must be accounted for when searching for potential off-target sites. In the case of SpCas9, Cas-OFFinder first compiles all the 23-bp DNA sequences composed of 20-bp sequences corresponding to the sgRNA sequence of interest and the 5′-NRG-3′ PAM sequences (Fig. 1A). Cas-OFFinder then compares all the compiled sequences with the query sequence and counts the number of mismatched bases in the 20-bp sgRNA sequence.
Fig. 1.

(A) The scheme of Cas-OFFinder. (B) The workflow of Cas-OFFinder. (C) Running time per target site as a function of the number of input target sites via CPU (black squares) and GPU (red circles)

The rpoC gene (RpoC, accession no.:
NP_268496.1) coding for N-terminal residues Phe7-Asp818 was amplified
by polymerase chain reaction (PCR) from the genomic DNA of the S. pyogenes M1 strain (MTCC) and cloned in the pEC-K-HT-HIS
(2) in-house vector at EcoR1 and BamH1 sites. The construct was transformed
into the expression host BL21 (DE3) strain of Escherichia
coli. For protein expression, the transformed E.
coli cells were cultured in Luria–Bertani (LB) medium.
An overnight culture (10 mL) was prepared and transferred to 1 L of
LB medium supplemented with 50 mg/mL kanamycin. The culture was grown
at 37 °C at 150 rpm shaking until the desired optical density
of 0.6 at A₆₀₀ was obtained. The culture was induced with
1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for
protein expression and incubated further for 4 h with shaking. The
cells were harvested by centrifugation at 4000 rpm for 20 min at 4
°C. The cell pellet was suspended in lysis buffer containing
20 mM Tris pH 7.0, 300 mM NaCl, and 10% glycerol, and the cells were
lysed by sonication on ice. The cell lysate was centrifuged at 10,000
rpm for 45 min at 4 °C, and the pellet was used for purification.
The protein was solubilized from the pellet using buffer containing
20 mM Tris pH 7.0, 300 mM NaCl, 10% glycerol, and 10% N-lauroylsarcosine by continuous stirring at 150 rpm for 1 h at 4
°C and centrifuged at 10,000 rpm for 30 min at 4 °C. The
solubilized protein was further refolded by stepwise membrane dialysis
against lysis buffer containing 5, 2.5, 1, and 0% N-lauroylsarcosine, respectively, for 3 h at 4 °C. As the protein
was expressed with an N-terminal His-tag, purification was carried
out by nickel-affinity chromatography. At each step, the purity of
the fractions was analyzed on a 10% SDS-PAGE gel. The concentration
of protein was measured using a UV spectrophotometer (A₂₈₀) and presumed calculated absorption coefficient of 0.789 from the
PROTPARAM online tool.²⁹