Serine Integrases: Advancing Synthetic Biology

Abstract

Serine integrases catalyze precise rearrangement of DNA through site-specific recombination of small sequences of DNA called attachment (att) sites. Unlike other site-specific recombinases, the recombination reaction driven by serine integrases is highly directional and can only be reversed in the presence of an accessory protein called a recombination directionality factor (RDF). The ability to control reaction directionality has led to the development of serine integrases as tools for controlled rearrangement and modification of DNA in synthetic biology, gene therapy, and biotechnology. This review discusses recent advances in serine integrase technologies focusing on their applications in genome engineering, DNA assembly, and logic and data storage devices.

Imaging cell lineage with a synthetic digital recording system

“…They mediate irreversible recombination between directional attP and attB target sites, deleting or inverting the intervening sequence depending on relative site orientation ( 28 – 32 ). Serine integrases such as Bxb1 do not rely on endogenous repair mechanisms to generate edits, and they function across species, including in mammalian cells ( 32 – 34 ). To create a trit, we flanked a barcode sequence by an inverted pair of attP sites on one end and an attB site on the other such that Bxb1-mediated recombination produces either irreversible barcode deletion or inversion (Fig.…”

Addressable, “Packet-Based” Intercellular Communication through Plasmid Conjugation

“…A three-node linear path can be implemented using only two gRNAs and one integrase (Fig. 5a)-for a message traveling from strain A to B to C, its address initially contains the binding site for the C gRNA, preventing direct transfer of the message from strain A to strain C. Strain B expresses an integrase and a cassette containing the binding site for the A gRNA, such that once the message plasmid is sent to a cell in the B strain, integrase-mediated cassette exchange [13] swaps the binding site on the message plasmid from the C binding site to the A binding site. Once this occurs, the message plasmid can no longer return to strain A, but can now be sent to strain C. The linear path is generalizable to any n-strain population and can be implemented using n-1 orthogonal gRNAs and n-2 orthogonal integrases for n ≥ 4 (Fig.…”

“…The fact that the address region is encoded on the message plasmids also allows the cells themselves to alter its content. By flanking the gRNA binding sites in the address region with integrase attachment sites, it is possible for the cells to express the corresponding integrases to modularly insert, remove, and swap binding sites from the address region, dynamically updating the recipient list for their messages [13]. The expression of these integrases could be regulated by signaling within the population circuit itself, enabling feedback-driven autonomous regulation of information flow within a population.…”

A reversible memory switch for plant synthetic biology based on the phage PhiC31 integration system

“…While this system provides great spatiotemporal resolution of gene induction, its adaptation to full plants is challenging due to strict light requirements as well as limitations performing long-term and inheritable memory storage. Serine integrases are powerful tools that can induce a stable change in the DNA and have extensively been used for many applications, including the design of toggle switches for bacteria and mammalian cells (40). We adapted the previously described bacterial switch by Bonnet et al, 2012, which was based on the Bxb1 phage integration system (28).…”