Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9.
The RNA-guided endonuclease Cas9 from Streptococcus pyogenes (SpCas9) forms the
core of a powerful genome editing technology. DNA cleavage by SpCas9 is
dependent on the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) in the
target DNA, restricting the choice of targetable sequences. To address this
limitation, artificial SpCas9 variants with altered PAM specificities have
recently been developed. Here we report crystal structures of the VQR, EQR, and
VRER SpCas9 variants bound to target DNAs containing their preferred PAM
sequences. The structures reveal that the non-canonical PAMs are recognized by
an induced fit mechanism. Besides mediating sequence-specific base recognition,
the amino acid substitutions introduced in the SpCas9 variants facilitate
conformational remodeling of the PAM region of the bound DNA. Guided by the
structural data, we engineered a SpCas9 variant that specifically recognizes
NAAG PAMs. Taken together, these studies inform further development of
Cas9-based genome editing tools.