An experimental census of retrons for DNA production and genome editing.

Retrons are bacterial immune systems that use reverse-transcribed DNA (RT-DNA) to detect phage infection. They are also deployed for genome editing, where they are modified so that the RT-DNA encodes an editing donor. Retrons are common in bacterial genomes, and thousands of unique retrons have been predicted bioinformatically. However, few have been characterized experimentally. We add to the corpus of experimentally studied retrons, finding 62 empirically determined, natural RT-DNAs that are not predictable from the retron sequence alone. We synthesize >100 previously untested retrons to identify the natural sequence of RT-DNA they produce, quantify their RT-DNA production and test the relative efficacy of editing using retron-derived donors to edit bacterial, phage and human genomes. We observe large diversity in RT-DNA production and editing rates across retrons, finding that top-performing editors are drawn from a subset of the retron phylogeny and outperform those used in previous studies, reaching precise editing rates of up to 40% in human cells.

Continuous multiplexed phage genome editing using recombitrons.

Bacteriophage genome editing can enhance the efficacy of phages to eliminate pathogenic bacteria in patients and in the environment. However, current methods for editing phage genomes require laborious screening, counterselection or in vitro construction of modified genomes. Here, we present a scalable approach that uses modified bacterial retrons called recombitrons to generate recombineering donor DNA paired with single-stranded binding and annealing proteins for integration into phage genomes. This system can efficiently create genome modifications in multiple phages without the need for counterselection. The approach also supports larger insertions and deletions, which can be combined with simultaneous counterselection for >99% efficiency. Moreover, we show that the process is continuous, with more edits accumulating the longer the phage is cultured with the host, and multiplexable. We install up to five distinct mutations on a single lambda phage genome without counterselection in only a few hours of hands-on time and identify a residue-level epistatic interaction in the T7 gp17 tail fiber.

Simultaneous multi-site editing of individual genomes using retron arrays.

During recent years, the use of libraries-scale genomic manipulations scaffolded on CRISPR guide RNAs have been transformative. However, these existing approaches are typically multiplexed across genomes. Unfortunately, building cells with multiple, nonadjacent precise mutations remains a laborious cycle of editing, isolating an edited cell and editing again. The use of bacterial retrons can overcome this limitation. Retrons are genetic systems composed of a reverse transcriptase and a noncoding RNA that contains an multicopy single-stranded DNA, which is reverse transcribed to produce multiple copies of single-stranded DNA. Here we describe a technology-termed a multitron-for precisely modifying multiple sites on a single genome simultaneously using retron arrays, in which multiple donor-encoding DNAs are produced from a single transcript. The multitron architecture is compatible with both recombineering in prokaryotic cells and CRISPR editing in eukaryotic cells. We demonstrate applications for this approach in molecular recording, genetic element minimization and metabolic engineering.

An experimental census of retrons for DNA production and genome editing.

Retrons are bacterial immune systems that use reverse transcribed DNA as a detector of phage infection. They are also increasingly deployed as a component of biotechnology. For genome editing, for instance, retrons are modified so that the reverse transcribed DNA (RT-DNA) encodes an editing donor. Retrons are commonly found in bacterial genomes; thousands of unique retrons have now been predicted bioinformatically. However, only a small number have been characterized experimentally. Here, we add substantially to the corpus of experimentally studied retrons. We synthesized >100 previously untested retrons to identify the natural sequence of RT-DNA they produce, quantify their RT-DNA production, and test the relative efficacy of editing using retron-derived donors to edit bacterial genomes, phage genomes, and human genomes. We add 62 new empirically determined, natural RT-DNAs, which are not predictable from the retron sequence alone. We report a large diversity in RT-DNA production and editing rates across retrons, finding that top performing editors outperform those used in previous studies, and are drawn from a subset of the retron phylogeny.

Simultaneous multi-site editing of individual genomes using retron arrays.

Our understanding of genomics is limited by the scale of our genomic technologies. While libraries of genomic manipulations scaffolded on CRISPR gRNAs have been transformative, these existing approaches are typically multiplexed across genomes. Yet much of the complexity of real genomes is encoded within a genome across sites. Unfortunately, building cells with multiple, non-adjacent precise mutations remains a laborious cycle of editing, isolating an edited cell, and editing again. Here, we describe a technology for precisely modifying multiple sites on a single genome simultaneously. This technology - termed a multitron - is built from a heavily modified retron, in which multiple donor-encoding msds are produced from a single transcript. The multitron architecture is compatible with both recombineering in prokaryotic cells and CRISPR editing in eukaryotic cells. We demonstrate applications for this approach in molecular recording, genetic element minimization, and metabolic engineering.