How is the Bridge recombinase mechanism better than CRISPR? Photo credit: Braňo via Unsplash
Genetic engineering, a buzz word in today’s media, refers to the modification of genes within an organism’s DNA to introduce desirable characteristics. For example, uses in agriculture may include generating genetically modified crops to be resistant to pesticides, produce more fruit, or improved nutritional content. One method of genetic engineering is called CRISPR, short for “Clustered Regularly Interspaced Short Palindromic Repeats”. Scientists developed CRISPR editing by mimicking a bacterial, viral defence system that uses specific enzymes to cut invading viral DNA and insert it into its own genome.
In CRISPR, scientists manipulate this mechanism by inserting a single strand of RNA in place of the viral DNA. RNA is a single-stranded transportable copy of DNA, which encodes the location and type of genetic edit into the cell along with the bacterial editing enzymes. In bacteria, these enzymes work by cutting the host DNA to enable the addition of new foreign DNA encoded by the RNA. Once the new DNA is inserted, the cell’s DNA repair machinery fixes the breaks, thereby permanently fixing the edit in the genome. CRISPR editing, however, is limited to only removing or introducing several bases, and often, DNA repair machinery can either remove the edit completely or introduce unwanted mutations, making it not very reliable.
To improve the potential of genetic engineering, scientists have been working on introducing larger pieces of DNA and eliminating errors associated with relying on the cell’s DNA repair machinery. To do this, they have once again turned to bacteria and discovered a novel biology called the ‘Bridge Recombinase’ that can be used to improve genetic engineering.
Like humans, bacteria contain long DNA sequences that “jump” to different locations within the genome; these pieces of DNA are called transposons. Transposons function to jumble up unused DNA regions to evolve new genes potentially. Transposon integration into the genome relies on enzymes called ‘recombinases’, which facilitate exchange. DNA exchange between two specific locations, the target and donor sequence, without breaking the DNA itself.
Transposons function to jumble up unused DNA regions to evolve new genes potentially.
Thousands of recombinases have been discovered, each able to target defined sequences and coordinate unique edits. This recombinase toolbox expanded genetic engineering but, unlike CRISPR, didn’t allow scientists to make unnatural edits within the genome. In a recent paper, Durrant et al, identified a family of recombinase enzymes called the ‘IS110 elements’, which use RNA, like CRISPR, to mediate the location and type of edits made by transposon movement, which could make these unnatural edits possible.
The RNA associated with ‘IS110 elements’ has a two-loop structure called ‘bridge RNA’ (bRNA). Through complementary base pairing, one loop of the bRNA ‘sticks’ to the target DNA sequence, and the other loop binds to the donor DNA sequence to be inserted in place of the target. The recombinase enzyme then orchestrates the transfer of the target and donor DNA, or transposons, in a complicated four-armed DNA structure, which ensures the DNA is never broken. By avoiding breaking the DNA, ‘IS110 elements’ prevent the need to use the host cell DNA repair machinery, making the transfer more efficient and accurate.
Through complementary base pairing, one loop of the bRNA ‘sticks’ to the target DNA sequence, and the other loop binds to the donor DNA sequence to be inserted in place of the target.
To reprogram the bridge recombinase mechanism for genetic engineering, Durrant et al. inserted plasmids, small closed-circle pieces of DNA that are passed from bacteria to bacteria, into E.coli. The plasmids used by Durrant et al. are transcribed by the E.coli, producing two types of RNA: the bRNA and messenger RNA, which is then translated to generate the required recombinase enzymes (Figure 1). The genome from the genetically engineered E.coli was extracted and sequenced to identify the location of the inserted donor DNA compared to the desired target. They discovered that most insertions happened right at the intended target site, showing that the bRNA helped guide the recombinase effectively. In this case, the inserted donor DNA encoded antibiotic resistance, which was observed to be inherited, demonstrating permanent genome integration.
Figure 1: Mechanism of bridge recombinase genetic engineering.
Left to right: The circle represents plasmid DNA; the grey section encodes recombinase enzymes, which are shown as grey dumbbells, and the orange/red section corresponds to bRNA. The orange section of bRNA recognises the target sequence (blue) within the bacteria’s genome, whereas the red binds the donor sequence (green). This positions the recombinase enzyme in place to facilitate the crossover of the donor to target DNA. Generating edited DNA is observed on the right.
The development of bridge recombinase technology could enable programmable large-scale DNA rearrangements, including insertions, inversions and deletions. Bridge recombinase editing could be used to replace damaged DNA associated with cancer or replace genes associated with hereditary disorders, which otherwise would require several low-efficacy CRISPR edits. Wider applications include genetic engineering in agriculture, antibiotic resistance, and, of course, answering fundamental questions in biological research.
Bridge recombinase editing could be used to replace damaged DNA associated with cancer or replace genes associated with hereditary disorders…
However, with all good things, there are limitations, and those of bridge recombinases aren’t insignificant. The main limitation is that this technique has only been performed on prokaryotic E. coli, a simple-celled organism without a nucleus, and not more complex eukaryotes that have, a nucleus and other membrane-bound organelles such as animals, plants and fungi. Researchers are optimistic, as CRISPR is a successful example of using RNA-programmable bacterial enzymes in eukaryotes. Another concern is that the short bRNA which binds specifically in the E.coli but may have off-target binding in the significantly larger eukaryotic genome. Durrant et al. are already experimenting with longer bRNAs designed to bind DNA more accurately and reduce unintended edits at similar sequences. Finally, the bridge recombinase technique shares a lot of limitations associated with CRISPR genetic engineering, including difficulty in delivery to many cells, low efficiency of cellular uptake, and removal of edits by host cell DNA repair machinery. Despite this, there is no doubt that bridge recombinase editing marks the dawn of the next generation of RNA-based genetic engineering, offering a vast range of potential for applications in academia and industry alike.
