Q: What is gene editing and what are the current challenges faced in the field?
A: Genome engineering tools such as CRISPR enable scientists to insert, delete, or edit DNA within organisms, effectively changing genes and phenotypes (characteristics) of those organisms. Although CRISPR is the most well-known of these genome engineering tools, it has several key limitations. This includes the requirement of cutting the genome, off-target effects and mistakes, and sequence requirements (which limits the sites that can actually be edited). Furthermore, larger changes such as genome insertions have very low efficiency and are difficult to achieve with tools like CRISPR.
Q: What is recombineering? Is that the same as CRISPR?
A: Lambda phage recombineering (or homologous recombination with bacteriophage proteins) is another genome engineering technique that can be used in E. coli. in order to avoid many of the issues faced by tools like CRISPR. Recombineering works by delivering a strand of ssDNA (single-stranded), which can be used to encode an insertion, deletion, or mutation of any kind. Single-stranded annealing proteins attach this ssDNA to the genome at the replication fork. Once the new DNA is attached to the genome, the new DNA is replicated. This effectively edits the genome. This process can be tailored to work at any desired location on the genome and does not require cutting of the genome. It is also extremely accurate and high-efficiency. It has enabled several new technologies like MAGE (“multiplexed automated genome engineering”) which allows researchers to successfully make many different edits simultaneously.
Q: What did you research in this paper?
A: There are many single-strand annealing proteins (that stick ssDNA to the replication fork) found in phages infecting different bacteria. We explored the specificity of those proteins, and found that compatible single-stranded binding (SSB) proteins are necessary for the SSAP mechanism (i.e. recombineering). By delivering pairs of compatible SSAPs and SSBs along with the ssDNA, we were able to perform edits at much higher efficiency in several different bacterial species. Notably, we were able to perform edits in one species using an SSAP-SSB pair from a different species, thus demonstrating the portability of SSAP-SSB systems.
Q: What is the significance of your findings?
A: While recombineering is simple, powerful, and versatile, prior to this work, it only worked well in E. coli. In this research, we were successful in generalizing the tool (i.e., recombineering and associated powerful methods like MAGE) to several other organisms and provided a framework for expanding its use even further.
We demonstrated the power of recombineering by exploring the fitness landscape of a gene involved in antibiotic resistance in L. lactis. We made millions of mutant variants of this gene, discovering tens of thousands of new changes conferring antibiotic resistance to the bacteria. Hopefully, our findings will facilitate exciting new applications and eventually enable high-efficiency recombineering across all species.
Q: How does this relate to your work at Keystone?
A: While Keystone obviously doesn’t participate in wet lab work (yet), at a fundamental level, all rigorous research is the same. The ultimate goal of research is to uncover truths, whether or not they are obvious truths. The work we do at Keystone emulates the work we did in lab: discovering interesting problems, building the frameworks to solve them, and putting in the work to see projects to their end.
To read the full paper click here.