The CRISPR-Cas approach, already valued as a means of accomplishing multiplex genome editing, promises to become increasingly versatile with the identification of additional CRISPR-Cas components. These components include a wider range of Cas9 enzymes, dual-RNA structures, and associated protospacer adjacent motifs (PAMs).
At present, RNA-guided Cas9 from Streptococcus pyogenes, S. thermophiles, and Neisseria meningitides have been developed into tools for genome manipulation. Now, according to researchers at the Helmholtz Centre for Infection Research (HZI), it may be possible to expand the possibilities of the RNA-programmable Cas9 toolbox to additional orthologous systems.
These researchers investigated the diversity and interchangeability of dual-RNA:Cas9 in eight representatives of phylogenetically defined type II CRISPR-Cas groups. So, besides introducing the prospect of additional CRISPR-Cas components, the researchers explored the evolutionary aspects of CRISPR-Cas systems, including coevolution and horizontal transfer of the system components.
The researchers published their results November 22 in Nucleic Acids Research, in an article entitled “Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems.” In this article, the authors wrote, “We identified and characterized dual-RNA and PAM requirements for eight Cas9 orthologous enzymes representative of the Cas9 phylogenetic grouping. To evaluate dual-RNA:Cas9 diversity, we performed bioinformatics analysis of type II CRISPR-Cas systems from available genomes and identified Cas9 orthologs in a plethora of bacterial species that belong to 12 phyla and were isolated from diverse environments.”
“This work,” continued the authors, “provides the first experimental evidence in support of tracrRNA:crRNA duplex and Cas9 protein coevolution.” The authors also considered the possibility that the various dual-RNA , Cas9 orthologs, and associated PAM sequences identified in their article may enhance CRISPR-Cas editing by offering “increased versatility and possibly specificity.”
One of the study authors, Emmanuelle Charpentier, Ph.D., head of HZI’s department of regulation in infection biology, after indicating that her group had analyzed and compared the enzyme Cas9 and the dual-tracrRNAs-crRNAs that guide this enzyme site-specifically to the DNA in various strains of bacteria, explained that her group’s findings allowed them the classification of the Cas9 proteins originating from different bacteria into groups. Within these groups, the CRISPR-Cas systems are exchangeable, which is not possible between different groups.
Materials provided by HZI suggest how the new results may alter laboratory practices: “The enzymes can be combined and thereby a variety of changes in the target DNA can be made at once.” New practical applications, too, may be on the horizon, including a new therapy for genetic disorders caused by different mutations in a patient’s DNA, according to HZI. “Furthermore, the method could be used to fight the AIDS virus HIV, which uses a receptor of the human immune cells to infect them. Using CRISPR-Cas, the gene for the receptor could be removed, and the patients could become immune to the virus.”
While cautioning that such applications may not be imminent, HZI still expresses optimism about the potential of the CRISPR-Cas technology. “Some of my colleagues already compare it to the PCR,” noted Dr. Charpentier.