Abstract
It has been anticipated that protospacer adjacent motif (PAM) specificity of the CRISPR-Cas9 nucleases protospacer appears to be modular. Here we present the finding that naturally occurring domain swapping has been extensively involved in altering its PAM specificities. Sequence analysis of streptococcal Cas9 sequences revealed conservation of three distinct PAM-interacting motifs, with phylogenetic analysis of full-length Cas9 and the PID demonstrating that PAM-interacting domain (PID) domain swapping was extensively utilized to diversify its PAM specificity. An extended analysis of 582 representative Cas9 sequences revealed that this PID swapping was broadly present in most of the investigated genera. Mimicking the natural PID domain swapping, a functional chimeric enzyme, based on the scaffold of compact Staphylococcus aureus Cas9, with novel NNAAAA PAM specificity was developed. In summary, our findings shed new light on a rich source of exchangeable PID domains in Cas9 genes, which can be mined for domain swapping aiming to an effective PAM refinement.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- crRNA
- CRISPR RNA
- DMEM
- Dulbecco’s modified Eagle’s medium
- PAM
- protospacer adjacent motif
- PID
- PAM-interacting domain
- RDP
- recombination detection program
- SpCas9
- Streptococcus pyogenes Cas9
- SpRY
- SpCas9 variant
- SaCas9
- Staphylococcus aureus Cas9
- SHyCas9
- Staphylococcus hyicus Cas9.