How Can We Defeat Superbugs with CRISPR/Cas9?

 

What if there were a way to cut HIV genes out of the human genome? What if we could get rid of genetic predisposition to diabetes? Will it ever be possible to eliminate the virulence of mono or destroy a crop’s ability to develop a disease?

It’s possible. Over time, it will be possible.

The up-and-coming tool for genetic modification is known in the synthetic biology community as “CRISPR/Cas9.”

Clustered regularly interspaced short palindromic repeats (conveniently abbreviated “CRISPR”) are DNA sequences that help defend bacteria against viruses.

The CRISPR/Cas9 system involves making double-stranded breaks in the DNA sequence of the gene to be modified. The system is found natively in bacteria, but scientists have found a way to utilize this natural mechanism as a tool for gene-specific modifications.

The Cas9 protein “captures” the region of DNA that needs to be modified (a.k.a. the “target gene”). It holds the DNA in place while a special type of RNA, called guide RNA (gRNA), binds to the target gene, and the Cas9 protein will cut this gene at a specific site. The specific cut site is dictated by the protospacer adjacent motif (PAM), which is Cas9’s way of distinguishing foreign DNA from native, or “genomic,” DNA.

Cas9 is remarkable for its ability to make double-stranded breaks on DNA. Cas9 will cut the sites of the target gene bound by gRNA, preparing the cut DNA for modification (such as deletions or insertions). To learn more about CRISPR/Cas9, check out this video from MIT, which does a great job of providing an explanation on how this system works.

 

 

Figure 1: The CRISPR/Cas9 System at Work

For our project, the DNA we want CRISPR/Cas9 to modify codes for antibiotic resistance.

Researchers have found that CRISPR/Cas9 can be used to successfully cleave antibiotic resistance in pathogenic bacteria. However, the mechanism for delivering a functional Cas9 protein to antibiotic-resistant bacteria is still being developed. Designing a delivery system for Cas9 is the goal of NU iGEM’s project.

DNA “spacer” sequences, situated between each of the “palindromic repeats,” are “records” of a bacteria’s encounters with substances--such as antibiotics--that are harmful to bacterial function. Bacterial genomes with these records become naturally selected and grow rampant over time, leading to strains of bacteria that are resistant against certain antibiotics. Hence, our fight against antibiotic-resistant superbugs is in full swing today.

As you can see in our project overview, we plan to use outer membrane vesicles (OMVs)--which are naturally secreted from Gram-negative bacteria--as the vehicle for protein delivery to pathogens. Our methods for generating OMVs are published here.

In short, the goal of the game is to have a strain of Gram-negative bacteria (in our case, E. coli) express a signal protein or protein complex (which are mechanisms that transport proteins through the inner membrane of E. coli and into the periplasm). Our E. coli strain also needs to overproduce OMVs that will encapsulate the Cas9 protein. The Cas9-filled OMVs can then be delivered to the pathogen, release the Cas9 protein, and allow the CRISPR/Cas9 system to delete a particular antibiotic-resistant gene*.

 

Figure 2: Delivery of Cas9 Through Inner Membrane via Signaling Sequence or Membrane Protein Pores

 

Figure 3: Cas9 Immersed in the Periplasm

 

Figure 4: Cas9 Diffusing Through Mesh Peptidoglycan Layer

 

Figure 5: Budding OMV Encapsulating Cas9

 

Figure 6: Complete OMV to be Delivered to Target Pathogen

Curious about what we do? Let’s talk!

*The particular antibiotic-resistant gene is specified through gRNA design. In other words, scientists can customize the gRNA to bind with different types of antibiotic resistance.