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.

A bacterial TIR-based immune system senses viral capsids to initiate defense.

Toll/interleukin-1 receptor (TIR) domains are present in immune systems that protect prokaryotes from viral (phage) attack. In response to infection, TIRs can produce a cyclic adenosine diphosphate-ribose (ADPR) signaling molecule, which activates an effector that depletes the host of the essential metabolite NAD+ to limit phage propagation. How bacterial TIRs recognize phage infection is not known.

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.

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.

Continuous Multiplexed Phage Genome Editing Using Recombitrons.

Bacteriophages, which naturally shape bacterial communities, can be co-opted as a biological technology to help eliminate pathogenic bacteria from our bodies and food supply. Phage genome editing is a critical tool to engineer more effective phage technologies. However, editing phage genomes has traditionally been a low efficiency process that requires laborious screening, counter selection, or construction of modified genomes.

Recording gene expression order in DNA by CRISPR addition of retron barcodes.

Biological processes depend on the differential expression of genes over time, but methods to make physical recordings of these processes are limited. Here we report a molecular system for making time-ordered recordings of transcriptional events into living genomes. We do this through engineered RNA barcodes, based on prokaryotic retrons, that are reverse transcribed into DNA and integrated into the genome using the CRISPR-Cas system.

Retron reverse transcriptase termination and phage defense are dependent on host RNase H1.

Retrons are bacterial retroelements that produce single-stranded, reverse-transcribed DNA (RT-DNA) that is a critical part of a newly discovered phage defense system. Short retron RT-DNAs are produced from larger, structured RNAs via a unique 2'-5' initiation and a mechanism for precise termination that is not yet understood. Interestingly, retron reverse transcriptases (RTs) typically lack an RNase H domain and, therefore, depend on endogenous RNase H1 to remove RNA templates from RT-DNA.