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From 2014.igem.org

Contents 1 Future Directions 1.1 Increase proficiency of phage packaging 1.2 Targeting intracellular pathogens 1.3 Multiple SNP targeting vectors 1.4 References:

Future Directions

Increase proficiency of phage packaging

Small levels of contamination will prevent any therapeutic agent for human use from being approved. In this experiment, small levels of helper phagemid were packaged into phage, which allows these phage to replicate inside a cell they infect. The helper phagemid was modified to further disrupt its packaging signal; however, this sequence overlaps necessary phage production genes so these attempts prevented the helper phagemid’s ability to produce phage. To address this issue, the next phase is to edit the genome of E. Coli to produce the structural proteins for phage. The resulting recombinant will be transformed with CRISPR-Cas9 phagemids to produce sequence specific, non-replicating phage. This eliminates the need for a helper phagemid, therefore eliminating unwanted, packageable plasmids.

Targeting intracellular pathogens

All pathogens have a phase in which they are exocellular where it is easy to deliver therapeutics. Unfortunately, patients rarely show symptoms for more serious infections, such as tuberculosis, until the infection is primarily intracellular. This poses a challenge, how to deliver drugs or phage to a pathogen that exists inside a mammalian cell. Display of ligands to a mammalian receptor, such as FGF2, have been shown to allow transduction of phage into mammalian cells¹. M13 genes can be modified to display ligands for mammalian uptake receptor to allow CRISPR-Cas phage to reach target pathogens in their intracellular environment.

Multiple SNP targeting vectors

During the course of investigation, it was discovered that surviving bacteria were often infected by recombinant phage whose spacer region was lost². Endogenous CRISPR regions contain multiple CRISPR elements coding for different spacers, each targeting a unique DNA segment from a pathogen. The addition of multiple spacers with unique target sites into BBa_K1445001 would increase the chance of retaining at least one spacer sequence, and allow this system to target multiple mutations within a species to ensure all pathogenic mutants are targeted. Using restriction digests and ligations, multiple spacers targeting pathogenic mutations in E. coli can be cloned onto the same phagemid and delivered with increased killing efficiency.

References:

1. LAROCCA, DAVID, PAUL D. KASSNER, ALISON WITTE, ROBERT LADNER, Andrew Baird, and GLENN F. PIERCE. "Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage." The FASEB Journal 13 (1999): 727-734. Print.

2. 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.