Retron

Bacteria face a challenge when DNA enters their cells by transformation, mating, or phage infection. Should they treat this DNA as an invasive foreigner and destroy it, or consider it one of their own and potentially benefit from incorporating new genes or alleles to gain useful functions? It is frequently stated that the short nucleotide sequence Chi (5' GCTGGTGG 3'), a hotspot of homologous genetic recombination recognized by Escherichia coli's RecBCD helicase-nuclease, allows E. coli to distinguish its DNA (self) from any other DNA (non-self) and to destroy non-self DNA, and that Chi is "over-represented" in the E...

First discovered in the 1980s, retrons are bacterial genetic elements consisting of a reverse transcriptase and a non-coding RNA (ncRNA). Retrons mediate antiphage defence in bacteria but their structure and defence mechanisms are unknown. Here, we investigate the Escherichia coli Ec86 retron and use cryo-electron microscopy to determine the structures of the Ec86 (3.1 Å) and cognate effector-bound Ec86 (2.5 Å) complexes. The Ec86 reverse transcriptase exhibits a characteristic right-hand-like fold consisting of finger, palm and thumb subdomains...

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 retrons1 , that are reverse transcribed into DNA and integrated into the genome using the CRISPR-Cas system2 . The unidirectional integration of barcodes by CRISPR integrases enables reconstruction of transcriptional event timing based on a physical record through simple, logical rules rather than relying on pretrained classifiers or post hoc inferential methods...

Retrons are prokaryotic genetic retroelements encoding a reverse transcriptase that produces multi-copy single-stranded DNA1 (msDNA). Despite decades of research on the biosynthesis of msDNA2 , the function and physiological roles of retrons have remained unknown. Here we show that Retron-Sen2 of Salmonella enterica serovar Typhimurium encodes an accessory toxin protein, STM14_4640, which we renamed as RcaT. RcaT is neutralized by the reverse transcriptase-msDNA antitoxin complex, and becomes active upon perturbation of msDNA biosynthesis...

Recombineering is an important tool in gene editing, enabling fast, precise and highly specific in vivo modification of microbial genomes. Oligonucleotide-mediated recombineering via the in vivo production of single-stranded DNA can overcome the limitations of traditional recombineering methods that rely on the exogenous delivery of editing templates. By modifying a previously reported plasmid-based system for fully in vivo single-stranded DNA recombineering, we demonstrate iterative editing of independent loci by utilizing a temperature-sensitive origin of replication for easy curing of the editing plasmid from recombinant cells...

Most bacteria and archaea possess multiple antiviral defence systems that protect against infection by phages, archaeal viruses and mobile genetic elements. Our understanding of the diversity of defence systems has increased greatly in the last few years, and many more systems likely await discovery. To identify defence-related genes, we recently developed the Prokaryotic Antiviral Defence LOCator (PADLOC) bioinformatics tool. To increase the accessibility of PADLOC, we describe here the PADLOC web server (freely available at https://padloc...

Canonical CRISPR-Cas9 genome editing technique has profoundly impacted the fields of plant biology, biotechnology, and crop improvement. Since non-homologous end joining (NHEJ) is usually considered to generate random indels, its high efficiency mutation is generally not pertinent to precise editing. Homology-directed repair (HDR) can mediate precise editing with supplied donor DNA, but it suffers from extreme low efficiency in higher plants. Therefore, precision editing in plants will be facilitated by the ability to predict NHEJ repair outcome and to improve HDR efficiency...

Retrons are bacterial genetic elements involved in anti-phage defense. They have the unique ability to reverse transcribe RNA into multicopy single-stranded DNA (msDNA) that remains covalently linked to their template RNA. Retrons coupled with CRISPR-Cas9 in yeast have been shown to improve the efficiency of precise genome editing via homology-directed repair (HDR). In human cells, HDR editing efficiency has been limited by challenges associated with delivering extracellular donor DNA encoding the desired mutation...

Exogenous DNA can be a template to precisely edit a cell's genome. However, the delivery of in vitro-produced DNA to target cells can be inefficient, and low abundance of template DNA may underlie the low rate of precise editing. One potential tool to produce template DNA inside cells is a retron, a bacterial retroelement involved in phage defense. However, little effort has been directed at optimizing retrons to produce designed sequences. Here, we identify modifications to the retron non-coding RNA (ncRNA) that result in more abundant reverse-transcribed DNA (RT-DNA)...

Reverse transcriptases (RTs) are found in different systems including group II introns, Diversity Generating Retroelements (DGRs), retrons, CRISPR-Cas systems, and Abortive Infection (Abi) systems in prokaryotes. Different classes of RTs can play different roles, such as template switching and mobility in group II introns, spacer acquisition in CRISPR-Cas systems, mutagenic retrohoming in DGRs, programmed cell suicide in Abi systems, and recently discovered phage defense in retrons. While some classes of RTs have been studied extensively, others remain to be characterized...

Genetic variation accelerates adaptation and resilience and enables the survival of species in their changing environment. Increasing the genetic diversity of crop species is essential to improve their yield and enhance food security. Synthetic directed evolution (SDE) employs localized sequence diversification (LSD) of gene sequence and selection pressure to evolve gene variants with better fitness, improved properties and desired phenotypes. Recently, CRISPR-Cas-dependent and -independent technologies have been applied for LSD to mediate synthetic evolution in diverse species, including plants...

The majority of previously described Staphylococcus aureus bacteriophages belong to three major groups, namely, P68-like podophages, Twort-like or K-like myophages, and a more diverse group of temperate siphophages. Here, we present the following three novel S. aureus "jumbo" phages: MarsHill, Madawaska, and Machias. These phages were isolated from swine production environments in the United States and represent a novel clade of S. aureus myophage. The average genome size for these phages is ∼269 kb with each genome encoding ∼263 predicted protein-coding genes...

Reverse transcriptases (RTs) catalyze the polymerization of DNA from an RNA template. These enzymes were first discovered in RNA tumor viruses in 1970, but it was not until 1989 that they were found in prokaryotes as a key component of retrons. Apart from RTs encoded by the 'selfish' mobile retroelements known as group II introns, prokaryotic RTs are extraordinarily diverse, but their function has remained elusive. However, recent studies have revealed that different lineages of prokaryotic RTs, including retrons, those associated with CRISPR-Cas systems, Abi-like RTs and other yet uncharacterized RTs, are key components of different lines of defense against phages and other mobile genetic elements...

Phage genome editing is crucial to uncover the molecular mechanisms of virus infection and to engineer bacteriophages with enhanced antibacterial properties. Phage genetic engineering relies mostly on homologous recombination (HR) assisted by the targeted elimination of wild-type phages by CRISPR-Cas nucleases. These strategies are often less effective in virulent bacteriophages with large genomes. T5 is a virulent phage that infects Escherichia coli . We found that CRISPR-Cas9 system (type II-A) had ununiform efficacies against T5, which impairs a reliable use of CRISPR-Cas-assisted counterselection in the gene editing of T5...

Bacteriophages or phages are viruses that infect bacterial cells (for the scope of this review we will also consider viruses that infect Archaea). The constant threat of phage infection is a major force that shapes evolution of microbial genomes. To withstand infection, bacteria had evolved numerous strategies to avoid recognition by phages or to directly interfere with phage propagation inside the cell. Classical molecular biology and genetic engineering had been deeply intertwined with the study of phages and host defenses...

Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications...

Bacterial retrons consist of a reverse transcriptase (RT) and a contiguous non-coding RNA (ncRNA) gene. One third of annotated retrons carry additional open reading frames (ORFs), the contribution and significance of which in retron biology remains to be determined. In this study we developed a computational pipeline for the systematic prediction of genes specifically associated with retron RTs based on a previously reported large dataset representative of the diversity of prokaryotic RTs. We found that retrons generally comprise a tripartite system composed of the ncRNA, the RT and an additional protein or RT-fused domain with diverse enzymatic functions...

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Retrons are bacterial genetic elements comprised of a reverse transcriptase (RT) and a non-coding RNA (ncRNA). The RT uses the ncRNA as template, generating a chimeric RNA/DNA molecule in which the RNA and DNA components are covalently linked. Although retrons were discovered three decades ago, their function remained unknown. We report that retrons function as anti-phage defense systems. The defensive unit is composed of three components: the RT, the ncRNA, and an effector protein. We examined multiple retron systems and show that they confer defense against a broad range of phages via abortive infection...

The Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to initiate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs), and confirmed these results by Cas9-mediated DSBs in combination with a bacterial retron system that produces ssDNA templates in vivo...