Team:CU-Boulder/Project/Motivation

From 2014.igem.org


Motivation


Since the discovery of Penicillin in 1928, antibiotics have been successfully used to treat bacterial infections. In the past several decades, bacteria have begun evolving resistances to the antibiotics. The emergence of carbapenem-resistant enterobacteriaceae has been declared a serious threat to public health by the CDC, while other pan-resistant species, such MRSA and resistant variants of tuberculosis still pose imminent risk to public health. Due to the ability of bacteria to share advantageous mutations through conjugation, antibiotic resistance is spreading rapidly between bacterial species and pressuring companies to produce new antibacterials. Unfortunately, antibiotics have a poor return on investment, encouraging many companies to abandon antibiotic research in favor of more profitable drugs. One such alternative to antibiotics is the less expensive and more adaptive treatment option of phage therapy. Whereas, antibiotics target conserved metabolic pathways that are shared by many bacterial species and results in damage to commensal bacteria in a patient, phage therapy can specifically target a species. But even this method does not provide the specificity to distinguish between individuals in a species.

A new therapeutic treatment for bacterial infections is necessary. A paper by Gomaa et al showed that a type I-E CRISPR-Cas could be used to specifically target and kill bacteria. The CRISPR-Cas system consists of two important parts: the Cas endonuclease and guide RNA (gRNA). The Cas endonuclease and CRISPR gRNA form a complex that searches genomic DNA for PAM motifs with upstream sequences that share a complementary region with the gRNA. If found, the Cas cleaves the DNA, resulting in a double stranded break. The gRNA is coded for by a spacer sequence that can be modified to direct the Cas protein to a different target sequence. Goma et al found that if this spacer sequence complimented the genome of the host cell adjacent to a PAM sequence, then the DNA would be targeted by the Cas, resulting in cell death. They then designed a spacer that would target one strain of bacteria but not another. Transforming the CRISPR-Cas machinery into a mixed population of both bacteria, they showed that the strain containing the target sequence was killed while the other strain was unharmed. Their experiment demonstrated the antibacterial potential of CRISPR-Cas9 but their experimental design lacked a delivery mechanism that could be used therapeutically.

To address the delivery problem posed by Gomaa et al, our experiment highlights the potential for non-replicating phage to be utilized as a delivery mechanism. During the laboratory experiment phase of this project, two papers were published in Nature Biotechnology that further demonstrate the potential of this system. Professor Marrafini of the Laboratory of Bacteriology at the Rockefeller University in New York┬▓ and Professor Timothy Lu of the MIT Microbiology Program at the Massachusetts Institute of Technology┬│ have demonstrated the specificity and effective delivery of CRISPR-Cas phage.


References:

1. Gomaa AA, Klumpe HE, Luo ML, Selle K, Barrangou R, Beisel CL. 2014. Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. mBio 5(1):e00928-13. doi:10.1128/mBio.00928-13.
2. David Bikard, Chad W Euler, Wenyan Jiang, Philip M Nussenzweig, Gregory W Goldberg, Xavier Duportet, Vincent A Fischetti, Luciano A Marraffini. 2014. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. doi:10.1038/nbt.3043
3. Robert J Citorik, Mark Mimee, Timothy K Lu. 2014. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. doi:10.1038/nbt.3011