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.

Temporally resolved transcriptional recording in E. coli DNA using a Retro-Cascorder.

Biological signals occur over time in living cells. Yet most current approaches to interrogate biology, particularly gene expression, use destructive techniques that quantify signals only at a single point in time. A recent technological advance, termed the Retro-Cascorder, overcomes this limitation by molecularly logging a record of gene expression events in a temporally organized genomic ledger. The Retro-Cascorder works by converting a transcriptional event into a DNA barcode using a retron reverse transcriptase and then storing that event in a unidirectionally expanding clustered regularly interspaced short palindromic repeats (CRISPR) array via acquisition by CRISPR-Cas integrases. This CRISPR array-based ledger of gene expression can be retrieved at a later point in time by sequencing. Here we describe an implementation of the Retro-Cascorder in which the relative timing of transcriptional events from multiple promoters of interest is recorded chronologically in Escherichia coli populations over multiple days. We detail the molecular components required for this technology, provide a step-by-step guide to generate the recording and retrieve the data by Illumina sequencing, and give instructions for how to use custom software to infer the relative transcriptional timing from the sequencing data. The example recording is generated in 2 d, preparation of sequencing libraries and sequencing can be accomplished in 2-3 d, and analysis of data takes up to several hours. This protocol can be implemented by someone familiar with basic bacterial culture, molecular biology and bioinformatics. Analysis can be minimally run on a personal computer.

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 supply1. 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 in vitro construction of modified genomes2. These requirements impose limitations on the type and throughput of phage modifications, which in turn limit our knowledge and potential for innovation. Here, we present a scalable approach for engineering phage genomes using recombitrons: modified bacterial retrons3 that generate recombineering donor DNA paired with single stranded binding and annealing proteins to integrate those donors into phage genomes. This system can efficiently create genome modifications in multiple phages without the need for counterselection. Moreover, the process is continuous, with edits accumulating in the phage genome the longer the phage is cultured with the host, and multiplexable, with different editing hosts contributing distinct mutations along the genome of a phage in a mixed culture. In lambda phage, as an example, recombitrons yield single-base substitutions at up to 99% efficiency and up to 5 distinct mutations installed on a single phage genome, all without counterselection and only a few hours of hands-on time.

UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions.

Reverse transcriptases (RTs) are enzymes capable of synthesizing DNA using RNA as a template. Within the last few years, a burst of research has led to the discovery of novel prokaryotic RTs with diverse antiviral properties, such as DRTs (Defense-associated RTs), which belong to the so-called group of unknown RTs (UG) and are closely related to the Abortive Infection system (Abi) RTs. In this work, we performed a systematic analysis of UG and Abi RTs, increasing the number of UG/Abi members up to 42 highly diverse groups, most of which are predicted to be functionally associated with other gene(s) or domain(s). Based on this information, we classified these systems into three major classes. In addition, we reveal that most of these groups are associated with defense functions and/or mobile genetic elements, and demonstrate the antiphage role of four novel groups. Besides, we highlight the presence of one of these systems in novel families of human gut viruses infecting members of the Bacteroidetes and Firmicutes phyla. This work lays the foundation for a comprehensive and unified understanding of these highly diverse RTs with enormous biotechnological potential.

Prokaryotic reverse transcriptases: from retroelements to specialized defense systems.

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. Prokaryotic RTs participate in various antiviral strategies, including abortive infection (Abi), in which the infected cell is induced to commit suicide to protect the host population, adaptive immunity, in which a memory of previous infection is used to build an efficient defense, and other as yet unidentified mechanisms. These prokaryotic enzymes are attracting considerable attention, both for use in cutting-edge technologies, such as genome editing, and as an emerging research topic. In this review, we discuss what is known about prokaryotic RTs, and the exciting evidence for their domestication from retroelements to create specialized defense systems.

Systematic prediction of genes functionally associated with bacterial retrons and classification of the encoded tripartite systems.

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. These retron systems are highly modular, and their components have coevolved to different extents. Based on the additional module, we classified retrons into 13 types, some of which include additional variants. Our findings provide a basis for future studies on the biological function of retrons and for expanding their biotechnological applications.

Recruitment of Reverse Transcriptase-Cas1 Fusion Proteins by Type VI-A CRISPR-Cas Systems.

Type VI CRISPR-Cas systems contain a single effector nuclease (Cas13) that exclusively targets single-stranded RNA. It remains unknown how these systems acquire spacers. It has been suggested that type VI systems with adaptation modules can acquire spacers from RNA bacteriophages, but sequence similarities suggest that spacers may provide immunity to DNA phages. We searched databases for Cas13 proteins with linked RTs. We identified two different type VI-A systems with adaptation modules including an RT-Cas1 fusion and Cas2 proteins. Phylogenetic reconstruction analyses revealed that these adaptation modules were recruited by different effector Cas13a proteins, possibly from RT-associated type III-D systems within the bacterial classes Alphaproteobacteria and Clostridia. These type VI-A systems are predicted to acquire spacers from RNA molecules, paving the way for future studies investigating their role in bacterial adaptive immunity and biotechnological applications.

Spacer acquisition from RNA mediated by a natural reverse transcriptase-Cas1 fusion protein associated with a type III-D CRISPR-Cas system in Vibrio vulnificus.

The association of reverse transcriptases (RTs) with CRISPR-Cas system has recently attracted interest because the RT activity appears to facilitate the RT-dependent acquisition of spacers from RNA molecules. However, our understanding of this spacer acquisition process remains limited. We characterized the in vivo acquisition of spacers mediated by an RT-Cas1 fusion protein linked to a type III-D system from Vibrio vulnificus strain YJ016, and showed that the adaptation module, consisting of the RT-Cas1 fusion, two different Cas2 proteins (A and B) and one of the two CRISPR arrays, was completely functional in a heterologous host. We found that mutations of the active site of the RT domain significantly decreased the acquisition of new spacers and showed that this RT-Cas1-associated adaptation module was able to incorporate spacers from RNA molecules into the CRISPR array. We demonstrated that the two Cas2 proteins of the adaptation module were required for spacer acquisition. Furthermore, we found that several sequence-specific features were required for the acquisition and integration of spacers derived from any region of the genome, with no bias along the 5'and 3'ends of coding sequences. This study provides new insight into the RT-Cas1 fusion protein-mediated acquisition of spacers from RNA molecules.

Multiple origins of reverse transcriptases linked to CRISPR-Cas systems.

Prokaryotic genomes harbour a plethora of uncharacterized reverse transcriptases (RTs). RTs phylogenetically related to those encoded by group-II introns have been found associated with type III CRISPR-Cas systems, adjacent or fused at the C-terminus to Cas1. It is thought that these RTs may have a relevant function in the CRISPR immune response mediating spacer acquisition from RNA molecules. The origin and relationships of these RTs and the ways in which the various protein domains evolved remain matters of debate. We carried out a large survey of annotated RTs in databases (198,760 sequences) and constructed a large dataset of unique representative sequences (9,141). The combined phylogenetic reconstruction and identification of the RTs and their various protein domains in the vicinity of CRISPR adaptation and effector modules revealed three different origins for these RTs, consistent with their emergence on multiple occasions: a larger group that have evolved from group-II intron RTs, and two minor lineages that may have arisen more recently from Retron/retron-like sequences and Abi-P2 RTs, the latter associated with type I-C systems. We also identified a particular group of RTs associated with CRISPR-cas loci in clade 12, fused C-terminally to an archaeo-eukaryotic primase (AEP), a protein domain (AE-Prim_S_like) forming a particular family within the AEP proper clade. Together, these data provide new insight into the evolution of CRISPR-Cas/RT systems.

On the Origin and Evolutionary Relationships of the Reverse Transcriptases Associated With Type III CRISPR-Cas Systems.

Reverse transcriptases (RTs) closely related to those encoded by group II introns but lacking the intron RNA structure have been found associated with type III clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, a prokaryotic immune system against invading viruses and foreign genetic elements. Two models have been proposed to explain the origin and evolutionary relationships of these RTs: (i) the "single point of origin" model, according to which these RTs originated from a single acquisition event in bacterial, with the various protein domains (RT, RT-Cas1, and Cas6-RT-Cas1 fusions) corresponding to single points in evolution; and (ii) the "various origins" model, according to which, independent acquisition events in different evolutionary episodes led to these fusions. We tested these alternative hypotheses, by analyzing and integrating published datasets of RT sequences associated with CRISPR-Cas systems and inferring phylogenetic trees by maximum likelihood (ML) methods. The RTs studied could be grouped into 13 clades, mostly in bacteria, in which they probably evolved. The various clades appear to form three independent lineages in bacteria and a recent lineage in archaea. Our data show that the Cas6 domain was acquired twice, independently, through RT-Cas1 fusion, in the bacterial lineages. Taken together, there more evidence to support the "various origins" hypothesis.

The Reverse Transcriptases Associated with CRISPR-Cas Systems.

CRISPR (clustered regularly interspaced short palindromic repeats) and associated proteins (Cas) act as adaptive immune systems in bacteria and archaea. Some CRISPR-Cas systems have been found to be associated with putative reverse transcriptases (RT), and an RT-Cas1 fusion associated with a type III-B system has been shown to acquire RNA spacers in vivo. Nevertheless, the origin and evolutionary relationships of these RTs and associated CRISPR-Cas systems remain largely unknown. We performed a comprehensive phylogenetic analysis of these RTs and associated Cas1 proteins, and classified their CRISPR-Cas modules. These systems were found predominantly in bacteria, and their presence in archaea may be due to a horizontal gene transfer event. These RTs cluster into 12 major clades essentially restricted to particular phyla, suggesting host-dependent functioning. The RTs and associated Cas1 proteins may have largely coevolved. They are, therefore, subject to the same selection pressures, which may have led to coadaptation within particular protein complexes. Furthermore, our results indicate that the association of an RT with a CRISPR-Cas system has occurred on multiple occasions during evolution.

Gestión de riesgos laborales en proyectos de rehabilitación ambiental de zonas contaminadas con hidrocarburos / Management of work risk factors in environmental rehabilitation projects of hydrocarbons polluted zones

La Estrategia Ambiental Cubana identifica la contaminación por residuos petrolizados como un problema ambiental. Dichos residuales presentan características particulares de manejo y se gestionan en el país a partir de la ejecución de proyectos de rehabilitación ambiental. El propósito de la investigación fue gestionar los factores de riesgos laborales existentes en los proyectos de rehabilitación ambiental de zonas contaminadas con hidrocarburos. Se seleccionó como caso de estudio el proyecto Rehabilitación ambiental integral de Punta Majagua, Cienfuegos, Cuba. Para lograr el propósito, se implementó un procedimiento utilizando la convergencia metodológica de diversas herramientas. Se combinaron técnicas de gestión de procesos, calidad y riesgos laborales, además de otros instrumentos de recopilación de información. Se identificaron 50 factores de riesgos laborales asociados a todos los procesos, donde el 40 por ciento de ellos fueron evaluados de críticos. Los procesos más peligrosos fueron la campaña de biorremediación y la creación de capacidades técnicas y logísticas. Se determinaron indicadores que demostraron la eficacia y efectividad de la estrategia de mitigación de riesgos. El estado de satisfacción laboral de los trabajadores después de la intervención fue evaluado de positivo, excepto las dimensiones de condiciones físicas de trabajo y salario . Los resultados corroboran la acertada gestión de riesgos laborales en el proyecto de estudio y permiten la generalización de las experiencias para obtener estándares de trabajo seguro en proyectos futuros, contribuyendo de esta manera al logro de producciones más limpias(AU)

The present Cuban Environmental Strategy identifies the contamination of crude oil wastes as a problem with particular managemental characteristics, which is managed by the implementation of environmental rehabilitation projects. The goal of this search was to manage the working risk factors dealt with the rehabilitation projects in hydrocarbon contaminated areas. The project Comprehensive environmental rehabilitation in Punta Majagua, Cienfuegos, Cuba was selected as a case study. To achieve this goal, a procedure using the methodological convergence of diverse tools was implemented. Different techniques of managemental processes, quality and working risks were used as well as several instruments for gathering information. 50 working risk factors were identified associated to all processes and 40 percent of them were assessed as critical. The most dangerous processes were the bioremediation campaign and the creation of technical and logistic capacities. Some indicators that revealed the efficacy and efficiency of the strategy in risk's mitigation were identified. The workers' satisfaction state at the workplace after this intervention was assessed as positive, except for the dimensions of physical working conditions and salary. These results confirm the right management of risk factors in this project as its possibility of generalizing experience to get safe working standards thus contributing to the achievement of clean productions(AU)