Advances in Genetic Engineering at the 2014 American Society for Microbiology Meeting

By Bio-IT World Staff 

May 22, 2014 | The annual meeting of the American Society for Microbiology (ASM) came to a close this Tuesday after three days of scientific sessions in Boston. Presentations at the event showed that rapid advances in the methods and reach of genetic engineering, which have been felt across the life sciences in the past year, are causing a particular stir in microbiology labs. Bacteria, with their small, nucleus-free genomes and open trade in plasmids, have traditionally been a first port of call for gene editing, and the talks at ASM provide an early look at the latest developments in the field.

The technology making the biggest impact this year has been CRISPR, which over a matter of months in late 2012 and early 2013 went from being a curiosity of the bacterial immune system to the most promising and adaptable new tool in the genetic engineer’s toolkit. The CRISPR system has allowed scientists to combine “guide RNA” sequences that match a specific stretch of DNA in a target genome, with a Cas9 protein that cuts both strands of DNA at that locus. This offers an unprecedented ability to easily remove, add, or modify selected sequences of DNA. In his ASM presentation on Tuesday, Stanley Qi of the UCSF Center for Systems and Synthetic Biology said simply, “Cas9 is magical.” (Bio-IT World has published a more detailed description of the CRISPR system in “Gene Therapy's Next Generation.”) 

Qi had come to ASM to present a re-engineered version of the Cas9 protein in which the nuclease, the domain that cuts DNA, has been made inactive. This “nuclease-deficient Cas9,” or dCas9, still binds to the genome at the targeted site, but instead of cleaving the DNA, it blocks transcription of the gene where it sits. Like RNA interference, or RNAi, this “CRISPRi” system can be used to silence genes one by one.

CRISPRi can also be made reversible, by combining dCas9 with an inducible promoter. In the presence of an inducer, the target genes are silenced, while removing the inducer restores partial gene transcription. Qi also suggested that dCas9 could be useful for labeling DNA, if combined with fluorescent proteins, or for modifying the genome at specific loci without carving out the native sequence.

Qi shared one application of CRISPRi, an extensive partial knockdown study of essential genes in Bacillus subtilis. By exposing B. subtilis to different combinations of CRISPRi and 35 different antibiotics, Qi’s lab was able to identify genes that contribute to antibiotic resistance. CRISPRi is useful for this kind of project because it is relatively simple to achieve partial suppression of essential genes, which does not kill the bacterium outright but may increase susceptibility to certain antibiotics, and because guide RNAs can be engineered very rapidly to target dCas9 to different genes. Qi and his colleagues even extended the project to knockdown combinations, in which pairs of genes were suppressed together, a reflection of how quickly and easily CRISPRi can be scaled up.

Stephanie Yaung, of George Church's lab at the Wyss Institute for Biologically Inspired Engineering, demonstrated a newly-discovered property of CRISPR that further distinguishes it from other gene editing methods like zinc finger nucleases or TALEN. All these technologies can be targeted to specific sequences in the genome, but most can only be guided to the four traditional, unmodified DNA bases. Methylated bases have proven a greater challenge to target.

To see if the same was true of CRISPR, Yaung and her colleagues tried to infect CRISPR-protected strains of E. coli with bacteriophages whose genetic payloads included methylated bases, including 5-hmC. Although the CRISPR defense systems specifically guided Cas9 to viral genes that included the methylated bases, Cas9 was still able to cut the viral genomes at the targeted sites. This suggests that modified bases will not deter Cas9, a finding with important implications for gene editing in mammalian cells, which tend to be much more heavily methylated than bacteria.

Daunting Challenges 

Gene editing may not be the only field where CRISPR eventually becomes a widespread technology. Mark Mimee, from Timothy Lu's Synthetic Biology Group at MIT, described the early stages of an intriguing effort to use CRISPR as a narrow-spectrum antibiotic. The idea makes a certain amount of intuitive sense: in nature, bacteria use CRISPR as an antiviral, and the ability to direct Cas9 to specific DNA sequences suggests it could be highly discriminating in attacking only selected pathogens.

The prospect is also highly appealing, given the scientific community’s growing alarm at the problem of antibiotic resistance, and calls for new narrow-spectrum drugs. Said Mimee, “[we are] asking the question, can we make a programmable-spectrum antimicrobial?... Can we get a list of antibiotic resistance genes, of virulence genes, of genes we consider bad, and make an antimicrobial to selectively find these cells, kill these, and spare all other cells in a population?”

In initial experiments, Lu’s lab has chosen antibiotic resistance genes in E. coli as targets, and built guide RNAs to direct Cas9 to these genes. Using phagemids to deliver the CRISPR system to mixed populations of E. coli, the group has found that it can drastically reduce the prevalence of only those strains carrying the chosen antibiotic resistance genes. The system used is not modified as an antibiotic; rather, the presence of the CRISPR system itself seems to cause cell death, an effect the group hypothesizes is due to native toxin-antitoxin systems in the bacterial genome that are destabilized by Cas9 activity. Mimee observed that the team was even able to kill E. coli when targeting genes located on plasmids, rather than the bacterial chromosome, and could also build CRISPR systems targeting two antibiotic resistance genes simultaneously.

The system is not a fully-functional antimicrobial – the team was never able to destroy the targeted populations entirely, and in the first in vivo experiments, on infected waxworms, an intervention with CRISPR-carrying phagemids only improved survival by 30%. Nevertheless, the notion of a “programmable-spectrum” antimicrobial is an exciting one, and one can hope that further experiments may increase the lethality of CRISPR without diminishing its specificity.

George Church was also present at ASM, to deliver one of his trademark whirlwind tours of cutting edge work among his wide circle of collaborators. On Tuesday, in a talk titled “Synthetic Ecologies,” his subject was the wholesale “re-coding” of E. coli genomes, removing entire basic elements of their genetic dictionaries.

Church, along with a variety of collaborators prominently including Farren Isaacs of Yale and Marc Lajoie at Harvard, has been working since at least 2011 to strip entire codons from a bacterial genome. This work began with a project to replace every TAG codon in E. coli with the synonymous TAA codon. TAG was chosen because it is the rarest codon in the organism’s genome, occurring in 314 locations, and because as a stop codon it may have been less essential to the cell than an amino acid-encoding sequence. The feat was achieved by inserting fragments that included TAG>TAA changes, 10 at a time, into 32 separate populations of E. coli. These populations were then paired up to exchange DNA, resulting in 16 populations with 20 changes each, and the process was repeated until all 314 changes coexisted in a single strain. As Church pointed out, this method of genetic engineering is so fast that the major time constraint is not how quickly new strains can be produced, but how quickly the researchers can check that their pairings have created the right genetic combinations. “We can make on the order of a billion genomes a day,” said Church. “We can screen, in this case, 100,000 genomes a day, so the bottleneck is the screening.”

This re-coding project was first published, short of completion, in 2011, but at ASM Church described major developments over the past year. The E. coli strain with no instances of the TAG codon is now complete and thriving. “It’s fully viable, and it’s resistant to two viruses that we’ve tested,” said Church, indicating one advantage of a genome-wide codon change. The incompatibility with natural organisms’ genomes confers viral resistance, and prevents the exchange of genetic material with naturally occurring bacteria.

More radically, the group has gone on to reintroduce TAG codons into re-coded E. coli strains, but change their meaning. With no TAG stop codons, the organism no longer relies on release factor 1 (RF1) – the protein that recognizes TAG as a stop codon – to accurately transcribe its DNA into RNA. The group therefore stripped RF1 from the bacterium, and instead introduced protein-tRNA complexes that read TAG as coding for two new amino acids that do not occur naturally. The new amino acids, p-azidophenylalanine and 2-naphthalalanine, were plugged into a gene for green fluorescent protein, which the organism successfully translated. The hope is to eventually use similar organisms to create useful proteins that could not be built using the natural repertoire of 20 amino acids.

Church reported that work on new re-coded E. coli strains is progressing steadily, most ambitiously an effort to remove every instance of 13 rare codons simultaneously from 42 essential genes, which has been partially successful.

The scale of genetic engineering challenges being tackled by ASM participants is remarkable, and shows how rapidly basic research in this field is moving. Microbiology is often viewed as a testing ground for gene editing techniques, but with increasingly sophisticated synthetic organisms, and new leads on antimicrobial discovery, the editing of bacterial genomes is also looking increasingly important in its own right.