ABOVE: Researchers in George Church’s lab modified wild type ADK proteins (left) in E.coli, furnishing them with an nonstandard amino acid (nsAA) meant to biocontain the resulting bacterial strain. CREDIT: AKOS NYERGES/HARVARD MEDICAL SCHOOL

At Harvard University’s Longwood campus, synthetic biologist and geneticist George Church finds himself busy. If he’s not guiding his students in building the latest biotech prototypes, he’s navigating new business ventures with his former ones.

Church is known for being a trailblazer in the bioengineering world. In some disciplines, he may be most popular for his work with direct genome sequencing and the Human Genome Project. In others, he and his students gained the spotlight by proving the capabilities of CRISPR-Cas9 editing in human cells. But the projects that his team is slogging away on now might have the next greatest impact on the scientific community at large.

“Our lab is sitting at an extreme point on the curve, and we’ve found that most times when we were out on the extreme, it's only a couple of years before we're overtaken by a tsunami of possibilities,” Church said.

This particular extreme point is multiplex genome editing. In his lab today, there are several unique use cases for genome-wide multiplex editing that synthetic biologists have their eyes on.

The multiplex toolbox

In the last few decades, gene editing has transitioned from a trope of science fiction to a pivotal part of reality. This advancement is largely due to the honing of CRISPR-based technologies, as well as other cutting-edge innovations. Rodolphe Barrangou, leader of the CRISPR lab at North Carolina State University, was one of the first to discover CRISPR’s initial repeated regions in the Streptococcus thermophilus genome. His group realized that the Cas9 enzyme led to a form of adaptive immunity; they showed how bacteria store, recognize, and share information about viral pathogens (1). CRISPR started as a natural way to screen for bacteriophage-resistant bacteria, Barrangou said in an email.

What Barrangou and others discovered from those humble beginnings led to many new capabilities for CRISPR editing, which is often described as the Cas9 enzyme guided by RNA that together snips away at a portion of DNA to either delete or insert new information. Today, CRISPR is capable of much more, including activating or repressing a gene using a deactivated form of a Cas protein — no snipping required. Other advances include base editing, which edits one base at a time using Cas proteins to make a small nick in one strand of the DNA (2).

Several cycles of MAGE can introduce edited segments of genetic information to multiple targets in a genome, as well as promote the growth of a diverse population of cells in a colony or swap out amino acids in polypeptide chains.

A big challenge still remains with gene editing, however, which is that scientists can only make a single edit here and there. Many diseases are polygenic in nature. And complex problems dealing with climate change and industrial bioengineering cannot be solved with one quick snip or by regulating one or two genes in an organism. For decades, the search continued to find a multigene editing technique or a suite of tools that can tackle complex polygenic changes.

As CRISPR took hold as the gene editing tool of choice, Church and his students already had their gears turning on how to scale up this and other novel biotech tools. In multiplex editing, the goal is to simultaneously modify more than one location in a genome (3). Preliminary research in devising methods to edit tens, hundreds, or even thousands of bases falls into this realm of multiplexing, but Church found that the biggest barrier for multigene edits is not efficacy; it’s safety. “It's a whole other thing to precisely edit unique sequences and make sure you don't have anything seriously off target,” Church said.

Off-target effects include improper phenotypic expression from nearby genes that were not modified or large-scale dysfunction of the genome after multiple edits. For example, as useful and efficient as CRISPR tech has been for editing anything from bacteria to humans, using its cleaving function repeatedly on a genome can be toxic to the organism, especially with therapeutics (3).

In 2009, Church and several of his former students developed a system called multiplex automated genomic engineering, or MAGE, that makes many edits without double-stranded breaks that can cause poor CRISPR outcomes. MAGE allows for diverse populations of cells to grow with genetic edits made by homologous recombination, the same phenomenon seen in meiosis, where nucleotide sequences cross over between similar chromosomes or genes. This editing technique is sometimes called "recombineering," and the bioinspired tool used early in MAGE systems comes from a lambda phage protein called Redβ, better known as the single-strand annealing protein, SSAP (4,5). 

While the CRISPR system is capable of editing genes in many different organisms, some genome engineering techniques were species dependent. SSAPs, for decades, were only used to modify bacteria such as Escherichia coli.The SSAP was found in a phage that easily infects E. coli and embeds its own genes into thegenome by harnessing the host’s binding proteins. For editing purposes, the SSAP attaches to an oligonucleotide that contains the edits. This strand of DNA synchs to the homologous location in unwound DNA, and the SSAP binds the strands together, starting at the lagging strand. This process is simple and analogous to Okazaki fragments seen during DNA replication (5,6).

SSAPs work with a host’s existing single-strand binding proteins to edit single-strand portions of DNA. Using SSAPs to bind edited genes during replication produces a new generation of edited cells, which can be scaled up using the MAGE system.

As part of the MAGE system, this process repeatedly occurs with many introductions of genetically edited oligonucleotides that match up with different parts of an E. coli genome. Then a technician introduces these edits to a colony of bacteria, where each microbe can incorporate and share these edits. Church said that a single researcher can facilitate making four billion diverse modified cells in a day using MAGE on dozens of sites (4).

"SSAPs are potentially more powerful than CRISPR in terms of their multiplex nature and their efficiency," said Charles Bell, a structural biologist at the Ohio State University's College of Medicine. "If we can engineer something that works in humans one day, I think MAGE could in some ways be more powerful."

Bell, who has collaborated with Church in the past, works closely with SSAPs and uses techniques such as cryogenic-electron microscopy to ascertain structures of these proteins. In a recent study, his team found a homologous protein to the lambda Redβ structure in a different phage, which also looks awfully similar to a protein found in humans called RAD52. The RAD52 protein is a part of a human DNA repair pathway, and it binds to single strands of DNA to anneal complementary strands (7).

"We can align the structures and show there is a common core fold, so our structures of the phage protein can help understand this overall mechanism," Bell said. “The structural work has strengthened the connection between the phage proteins and human RAD52.”

This finding may be the next step in developing multiplexable SSAPs for human use. But until then, Church and other researchers are coupling multiplexing with tried-and-true tools. For instance, Church’s team has shown that base editing works in a multiplex fashion by editing 33 essential genes in human stem cells (8). CRISPR editing can also work at a genome-wide scale — regardless of off-target effects — for the purpose of obtaining compounds needed in drug development such as producing the anticoagulant heparin from mammalian cells instead of extracting it from pigs (9). More research shows that SSAPs can aid in the double-strand break repair systems of CRISPR and reduce unwanted effects, potentially improving single-gene and multigene editing (10).

Barrangou believes that multiplex CRISPR editing can be safe and completely feasible for many applications, which has great upsides for biodesign, allowing the synthetic biology community to take the next leap in genomic editing. “Think ‘genome editing on steroids’…in the era of synthetic biology,” he said. For bioengineering, more multiplex use cases are popping up in curious places.

Safeguarding our microbial factories

The Church team has employed the MAGE system in a variety of special ways since its development. But the use of MAGE to defend bacteria from viruses is arguably one of the team’s most foundational research areas.

In 2013, Church and his students made a small tweak to the E. coli genome to modify how transfer RNA delivered and installed amino acids to a peptide chain. This initial "recoded" strain with more than 320 edits could synthesize its own proteins, no problem. But when a phage unwittingly entered, it could not replicate and infect its host. This was the first demonstration of resistance to viruses and even mobile genetic elements like plasmids (11).

In a recent study led by Akos Nyerges, a synthetic biologist in the Church lab, the team changed the genetic code of E. coli again. The even more buffed strain, called Ec_Syn61Δ3-SL, prevents phages from hijacking its genomic translational machinery to replicate viral proteins and has an additional safeguard (12).

Akos Nyerges uses MAGE to genetically protect E. coli from invading phages and prevent those modified genes from escaping into the environment.

Nyerges scanned an older version of the modified E. coli’s genome for some codons to recode using reprogrammed tRNAs. Two codons for the serine amino acid were reinstructed to produce leucine instead. And one stop codon was repurposed to introduce an amino acid not seen in any living systems, called a “nonstandard amino acid” or nsAA. All three of these changes across the genome kept Ec_Syn61Δ3-SL's life code stable while mistranslating any virus that entered, thus stopping a viral invasion in its tracks.

Compared to the earlier attempt almost a decade ago, this new modified E. coli showed resistance to all tested molecular invaders, which made it even more resistant to novel viruses. Nyerges' team discovered 12 new phage strains that readily infected the previous iteration of the modified E. coli but not the lab’s new modified strain. Nyerges isolated these phages everywhere from sewage to river water to agricultural soils that house animal grazers. "This [sampling] showed that the viral genome harbors numerous unexplored functions," Nyerges said. "These phages are phylogenetically similar to ones that people already found in industrial infections, but not specifically for lab-recoded organisms. So, some of the relatives of these phages might be problems in industrial fermentations and laboratory infections."

The team is ecstatic that this recoding created a secure "firewall" for infections but is especially thrilled to see that Ec_Syn61Δ3-SL was "biocontained" — not in the manner that laboratories and other facilities biocontain pathogens and other biohazards. This is biocontainment at the molecular level.

Most lab organisms are very fragile, very uncompetitive with the wild type versions, but you can imagine one that is healthy enough to be used in manufacturing and resistant to all viruses will have a huge advantage in the outside world. 
—George Church, Harvard University

Nyerges said that biocontainment in their genetically recoded cells is beneficial because they do not want the accidental escape of cells and the proliferation of altered genes – even when it is extremely unlikely to happen. The nsAA became the key security measure in case the new E. coli strain gets out into the wild (13). "If you make a multivirus resistant organism, it is one of the few lab organisms that could actually survive for more than a few minutes in the wild," Church said. "Most lab organisms are very fragile, very uncompetitive with the wild type versions, but you can imagine one that is healthy enough to be used in manufacturing and resistant to all viruses will have a huge advantage in the outside world."

Church likes to call out Michael Crichton's “Jurassic Park” both for attempting a form of biocontainment but also for failing to establish an accurate one. "It was called the 'lysine contingency' where the dinosaurs could not produce the amino acid lysine. But it's present in all foods; that's how we get this essential amino acid," Church said. "We want to take the ‘Jurassic Park’ approach, but we want to do it right."

Church said that it would take hundreds of changes for a phage to overcome the mass edit in Ec_Syn61Δ3-SL, so this is true protection. And the goal of this lab and others around the world will be to adapt this protection to sustainably produce recoded microorganisms for industries like bioremediation or biofuels (12), as well as for ambitious endeavors such as altering human genes for direct therapy (8).

Why mass editing has mass appeal

Virus-proofing and biocontaining modified E. coli was an important starting point, Nyerges said. “There is a very wide scope, either resistance or MAGE as a whole, that would be a huge plus for other fields,” he added.

Church said that this work translates extremely well into xenotransplantation, another big ticket study in his lab. Early in 2022, a 57-year-old man from Maryland received a pig heart transplant that extended his life for nearly two more months. It was not his immune system that rejected the transplant, which is usually the main concern. It was a herpesvirus found only in pigs that led to his demise (14). Church and his former student Luhan Yang, who now leads the biotech firm Qihan, founded eGenesis to take on these very xenotransplantation risks.

At eGenesis, Church and Yang targeted 62 copies of porcine-endogenous retroviruses (PERVs), which are viral elements embedded in the pig’s genome, using kidney cells. In pigs, PERVs are harmless; in humans, researchers are unsure how detrimental they may be. Church and Yang investigated these elements and used a multiplexed CRISPR-Cas9 system to inactivate the 62 copies of the viral genes, which reduced the transmission of the PERVs from pig cells to human cells by more than a thousand-fold (15). This proof of concept could lead to production of viable mammalian cells or transplants for therapeutics.

Like PERVs, humans have endogenous retroviruses and other repetitive elements peppered throughout our genomes. These repeated regions used to be viewed as “junk DNA” but now studies show that they can go as far as disrupting gene expression, initiating disease, and influencing the aging process (16). “We’re essentially looking at every category of major repeats in the human genome, some of which are millions of repeats,” Church said. His lab has gone as high as investigating 24,000 repetitive elements using multiplexing as the technique of choice.

Aside from biomedical applications, Church is taking multiplex engineering to newer frontiers, right off the silver screen. With his company Colossal, his researchers aim to de-extinct genes of mammoths and Tasmanian tigers, as well as protect one of the world’s largest ecosystem engineers: elephants. So, a world like that in “Jurassic Park” may not be so farfetched after all.

Even if multiplex genomic engineering doesn’t make waves the way CRISPR technology did, Church at least hopes that this new biotech can trigger a cascade of inspiration the way it has in his own lab. “Everything we have done to improve multiplex editing, even the bacterial multiplex editing, has influenced our work on the mammalian cells,” Church said. “And everything we do on human stem cells has benefitted the elephant project, and it goes on. There's some synergy between these fields by using multiplex.”

  1. Barrangou, R. et al. Genomic impact of CRISPR immunization against bacteriophages. Biochem Soc Trans 41, 1383-1391 (2013).
  2. Roberts, A. et al. Applications of CRISPR-Cas systems in lactic acid bacteria. FEMS Microbiology Reviews  44, 523-537 (2020).
  3. Thompson, D.B., et al. The Future of Multiplexed Eukaryotic Genome Engineering. ACS Chemical Biology  13, 313-325 (2017).
  4. Wang, H.H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature  460, 894-898 (2009).
  5. Wannier, T.M. et al. Recombineering and MAGE. Nat. Rev. Methods Primers  1, 7 (2021).
  6. Brewster, J.L. et al. Half a century of bacteriophage lambda recombinase: In vitro studies of lambda exonuclease and Red-beta annealase. IUBMB Life  72, 1622-1633 (2020).
  7. Caldwell, B.J. et al. Structure of Rad52 homolog from bacteriophage in complex with a novel duplex intermediate of DNA annealing. Preprint at: www.biorxiv.org/content/10.1101/2022.03.17.484533v1.full
  8. Chen, Y. et al. Multiplex base editing to convert TAG into TAA codons in the human genome. Nature Communications  13, 4482 (2022).
  9. Thacker, B.E. et al. Multiplex genome editing of mammalian cells for producing recombinant heparin. Metabolic Engineering  70, 155-165 (2022).
  10. Wang, C. et al. Microbial single-strand annealing proteins enable CRISPR gene-editing tools with improved knock-in efficiencies and reduced off-target effects. Nucleic Acids Research  49, 6 (2021).
  11. Lajoie, M.J. et al. Genomically Recoded Organisms Expand Biological Functions. Science  342, 357-360 (2013).
  12. Nyerges, A. et al. Swapped genetic code blocks viral infections and gene transfer. Preprint at: www.biorxiv.org/content/10.1101/2022.07.08.499367v1.full
  13. Mandell, D.J. et al. Biocontainment of genetically modified organisms by synthetic protein design. Nature  518, 55-60 (2015).
  14. Griffith, B.P. et al. Genetically Modified Porcine-to-Human Cardiac Xenotransplantation. N Engl J Med  387, 35-44 (2022).
  15. Yang, L. et al. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science  350, 1101-1104 (2015).
  16. Smith, C.J. et al. Enabling large-scale genome editing at repetitive elements by reducing DNA nicking. Nucleic Acids Research  48, 5183-5195 (2020).
Blue DDN

This story was originally published on Drug Discovery News, the leading news magazine for scientists in pharma and biotech.