Comparing Bacteriophage Recombineering of Electroporated DNA, CRISPR-Cas-Mediated Genome Engineering, and Yeast Based Assembly of Phage Genomes for Improving Phage Efficacy

Tommy W

Marin Academy Research Collaborative Program

Bacteriophages

Phage Overview:

Bacteriophages are viruses that infect, replicate in, and lyse only bacterial cells. Phages are constituted of a DNA or RNA genome enclosed in a protein capsid. Their components may include leglike appendages that the phage uses to bind to host cells and a central shaft through which the phage injects its genetic material into the host cell cytoplasm. Phages are extremely specific in the bacteria or archaea that they infect, generally being able to attach to only a certain species of bacteria, or in some instances only certain strains within a species. Bacteriophages identify and attach to specific receptors on the outside of a bacterium’s cell wall, which determine the phage host range. Once a phage has bound to a host cell, the sheath contracts and drives a tail-like tube through the cell wall, then it injects genetic material through a central shaft into the cytoplasm of the host cell. The virion is then replicated until the cell lyses and virions are spread to neighboring cells to repeat the process.

Phage Applications:

Due to phage lethality and specificity, they have a myriad of applications, especially as a potential solution to the problem that drug-resistant bacteria pose. Phages are vectors for horizontal gene transfer and can be used to introduce genes into specific strains of antibiotic-resistant bacteria (Pires et al., 2016). Phages are self-replicating within a host cell so if administered as an antibiotic, only one dose would be required to treat a bacterial infection. Additionally, they only target specific bacteria so the microbiome would remain undisturbed while the phages treated an infection.

Phage Limitations:

However, natural phages have limited efficacy. A phage that eliminates the entirety of its host population will enter starvation because phages depend upon the bacteria they prey on as a food source, and so must coexist in equilibrium with their bacterial food source. Natural selection favors moderately effective phages over highly effective phages in order to maintain predator-prey equilibrium (Huss & Raman, 2020).

CRISPR-Cas-Mediated Genome Engineering

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system is a prokaryotic immune defense against foreign genetic material. Recently adapted for genetic engineering, CRISPR-Cas systems effector complexes contain Cas (CRISPR associated) proteins and cr(CRISPR)RNA which bind to crRNA mediated target sequences. Once bound, the Cas protein creates a double-stranded break in the DNA. CRISPR-Cas systems can be used to modify, insert or delete genes within a phage genome. gRNA guides the effector complex to the target site of interest.

When introduced to a phage genome, the CRISPR system binds to and cleaves a specified target site determined by the gRNA. Mutated phages are then isolated and screened via PCR (Bari & Hatoum-Aslan, 2019). The CRISPR-Cas system allows for highly specific genomic editing. Both parts of the effector complex can be chosen from different CRISPR classifications to accomplish different purposes. CRISPR-Cas-based engineering is extremely intuitive, as synthetic plasmids can also be created as a vector for engineering.

All progeny created using this technique will bear the desired mutations (if there is no manual error), overcoming the issue of having to screen for mutant phages (Hupfeld et al., 2018). One limitation is that some natural phages have evolved anti-CRISPR immune defense mechanisms, which can make using this technique difficult.

Phage Engineering

Proposed Research:

Current phage engineering techniques being developed include Homologous Based Recombination, Rebooting Phages Using Assembled Genomic DNA, Bacteriophage Recombineering of Electroporated DNA (BRED), CRISPR-Cas-Mediated Genome Engineering, Whole Genome Synthesis Assembly from Synthetic Oligonucleotides, and Yeast Based Assembly of Phage Genomes are being developed to overcome this issue. Such techniques allow researchers to remove starvation-induced regulatory genes and engineer phages in other ways that can lead to efficiency increases of up to a thousandfold. I propose to compare the engineering techniques of BRED, CRISPR-Cas-Mediated Genome Engineering, and Yeast Based Assembly of Phage Genomes.

Bacteriophage Recombineering of Electroporated DNA

Bacteriophage Recombineering of Electroporated DNA (BRED) can be used to create point mutations, replace, insert or delete genes (Brown et al., 2017). Electroporation is the process of introducing genetic material into a cell using electrical pulses to briefly open cell membrane pores. BRED comprises the introduction of phage DNA and dsDNA into bacterial cells containing a plasmid that encodes proteins that promote homologous recombination (RecE protein) via coelectroporation (Pines et al., 2015).

The dsDNA is composed of the desired mutations buffered by regions of homology that align with the loci immediately above and below the desired target site for mutation in the phage genome. The host bacterial cell containing the dsDNA and phage DNA substrates are mixed with wild-type bacteria, plated, then monitored for the presence of phage plaques (Dunne et al., 2019). Plaques are then screened for the correctly mutated phage genome using PCR.

BRED allows for more specificity in mutations than homologous recombination, as using the technique researchers can insert, delete and replace genes. Phages can be obtained at high frequencies using BRED and mutants can be examined by a small number of polymerase chain reactions. However, BRED requires highly competent bacterial hosts in order to produce viable mutants. The technique may also result in the death of bacterial host cells. It also relies highly on co-transformation of the genetic substrates within the same cell meaning BRED is difficult to use in low transformation efficient Gram-positive bacteria (Kilcher & Loessner, 2019).

Yeast Based Assembly of Phage Genomes:

Phage genome propagation can lead to host cell death in bacteria, so Saccharomyces cerevisiae can be used as an intermediate host to undergo genetic manipulation. Phage genomes can be maintained easily in yeast. A phage genome is captured in a bacterial shuttle vector for S. cerevisiae containing homologous sequences at the ends of the phage genome (Kim et al., 2019). The vector and phage genome then join by homologous recombination. The phage genome is then captured and introduced to bacterial hosts to generate functional phages (Kilcher & Loessner, 2019).

A benefit of using yeast instead of bacteria as an intermediate host for genetic manipulation is that phages aren’t toxic to yeast cells. Genetic manipulation of phages in bacteria has limited efficiency because of the potential toxicity of the phage genome, but yeast can overcome this issue. This process is limited in its efficiency by bacterial transformation efficiencies because the technique requires phage genome extraction from the yeast, and introduction to bacteria (Grigonyte et al., 2020).

UCSD Center for Innovative Phage Applications and Therepeutics

IPATH, UCSD's center for phage research is the first dedicated phage therapy center in North America and is currently researching solutions using phages as a treatment for antibiotic-resistant infections.

FDA Emergency Approval of Phage Therapy for COVID-19 Patients

Bacterial infections as a complication of severe COVID-19 cases have left many patients who are already fighting off the SARS-CoV-2 virus dealing with antibiotic-resistant bacterial infections as well. The FDA has approved Adaptive Phage Therapeutics clinical phage therapy for use in these patients, with promising results.

Phages as Viral Vectors

Bacteriophages have potential as viral cloning vectors. As phages generally contain linear DNA, a single break in the DNA leads to two fragments that can be joined with foreign DNA to create a chimeric phage particle.

Signifigance

I hope to determine which of the phage engineering techniques that I propose to study are most effective in the context of creating transformed phages with greater lethality. This is important as convergent evolution with bacteria has led to inherent imposed restrictions on phage lethality. In order for the full therapeutic potential of phages to be harnessed, these restrictions must be removed with genetic engineering. In the future, as phage engineering develops, it is likely that it will also be feasible to modify the host-cell range of phages, allowing researchers to modify the extreme specificity of bacteriophages to lyse a particular pathogenic bacteria causing infection

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