New York, NY, February 05, 2019 --(PR.com
)-- Dr. Zhen Xie and Dr. Dacheng Ma published a research work titled, “Engineer chimeric Cas9 to expand PAM recognition based on evolutionary information.” In this study, a group of chimeric Cas9 (cCas9) variants were generated. This was done by “replacing the key region in the PAM interaction (PI) domain of Staphylococcus aureus Cas9 (SaCas9) with the corresponding region in a panel of SaCas9 orthologs.”
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) system is to function as the prokaryotic adaptive immune systems that prevent infection by providing protection. By repurposing, CRISPR/Cas system has shown revolutionized effect in biomedical research and application. Cas9 is one type of the nuclease in CRISPR system that can be directed to specific genome sites by a small RNA named guide RNA (gRNA). It is very useful for gene therapy to genetic disorder due to specific mutations. Several clinical trials have been launched.
However, short protospacer adjacent motif (PAM) is required downstream of the genome target site for Cas9 nuclease activity, hence, only sites with the correct PAM sequences could be ideally targeted which restrict the target range of this biomedical tool.
The 2018 chemistry Nobel prize
has been awarded to scientists developing the directed revolution of enzyme and protein.The study conducted by Xie and Ma developed an interesting approach for PAM expansion based on natural diverse of the CRISPR system. The CRISPR system has been found in half of all sequenced bacterial genomes. They found out that approximately all archaeal genomes, as well as CRISPR nucleases, were highly diverse.
The prime focus of the research by Xie and Ma is on Staphylococcus aureus (SaCas9) engineering. This is because SaCas9 is a compact Cas9 ortholog suitable for viral delivery or biomedical applications. In the study, the team of researchers developed a strategy to engineer SaCas9 variants with altered PAM recognition specificity.
Xie and Ma searched for SaCas9 orthologs in the NCBI database by performing a BLAST analysis with the full-length amino acid sequences of SaCas9. Further, 32 unique chimeric Cas9 variants were generated by replacing the PAM interaction region of the Cas9 orthologs. Xie, Ma and the team of researchers have provided a panel of cCas9 variants that are accessible up to a quarter of all the PAM sequences. They are inclusive of a compact size suitable for viral delivery in mammalian cells, which will be of great importance in the biomedical applications for the Cas9 positioning.
The study conducted by Xie and Ma showed the strategy, which was developed by them to engineer PAM recognition specificity. They performed it by swapping the key region in the PI domain in SaCas9 orthologs. According to them, directed evolution screening and structure-guided mutagenesis based on these cCas9 variants may further assist in improving the DNA cleavage activities. An explanation of the above can be that sufficient PAM binding activity of SaCas9 nucleases may be required to initiate strong gene editing activities. Furthermore, the explanation was also that relaxed PAM binding activity of SaCas9 nucleases activity was found to result in reduced DNA cleavage activity.
The researchers, however, feel that further studies are essential to understanding the functional relationship between the PAM recognition and the SaCas9 nuclease activity. After a couple of experiments and their analysis, one possible explanation was that the specific recognition and broad recognition contradicted each other due to the nature of protein-DNA interaction.
The research conducted by Xie and Ma is brilliant because it explains a quite complex subject in an easy manner with various experimental illustrations along with the results. It provides the reader with information that may be insightful to engineer Cas9 proteins for other purposes such as immugeneticity, high-fidelity and functional capability of mammalian cells.