Genetic Entanglement Enables Ultrastable Biocontainment in the Mammalian Gut.

Imbalances in the mammalian gut are associated with acute and chronic conditions, and using engineered probiotic strains to deliver synthetic constructs to treat them is a promising strategy. However, high rates of mutational escape and genetic instability limit the effectiveness of biocontainment circuits needed for safe and effective use. Here, we describe STALEMATE (equence enngd ulti lyered geneic buffring), a dual-layered failsafe biocontainment strategy that entangles genetic sequences to create pseudoessentiality and buffer against mutations.

Ku limits RNA-induced innate immunity to allow Alu expansion in primates.

Ku70 and Ku80 form the Ku heterodimer, a ring-shaped complex that initiates the non-homologous end-joining (NHEJ) DNA repair pathway. Ku binds to double-stranded DNA ends and recruits other NHEJ factors, including LIG4 and DNA-PKcs. Although Ku can bind to double-stranded RNA (dsRNA) and trap mutated DNA-PKcs on ribosomal RNA, the physiological role of the Ku-RNA interaction in otherwise wild-type cells remains unclear.

Ku suppresses RNA-mediated innate immune responses in human cells to accommodate primate-specific Alu expansion.

Ku70 and Ku80 form Ku, a ring-shaped protein that initiates the non-homologous end-joining (NHEJ) DNA repair pathway. Specifically, Ku binds to double-stranded DNA (dsDNA) ends and recruits other NHEJ factors ( , DNA-PKcs and LIG4). While Ku binds to double-stranded RNA (dsRNA) and traps mutated-DNA-PKcs on ribosomal RNA the physiological significance of Ku-dsRNA interactions in otherwise wild-type cells remains elusive.

Intein-based thermoregulated meganucleases for containment of genetic material.

Limiting the spread of synthetic genetic information outside of the intended use is essential for applications where biocontainment is critical. In particular, biocontainment of engineered probiotics and plasmids that are excreted from the mammalian gastrointestinal tract is needed to prevent escape and acquisition of genetic material that could confer a selective advantage to microbial communities. Here, we built a simple and lightweight biocontainment system that post-translationally activates a site-specific DNA endonuclease to degrade DNA at 18°C and not at higher temperatures.

Synthesis of a Conjugative System from Human Gut Metagenomic Data for Targeted Delivery of Cas9 Antimicrobials.

Metagenomic sequences represent an untapped source of genetic novelty, particularly for conjugative systems that could be used for plasmid-based delivery of Cas9-derived antimicrobial agents. However, unlocking the functional potential of conjugative systems purely from metagenomic sequences requires the identification of suitable candidate systems as starting scaffolds for DNA synthesis. Here, we developed a bioinformatics approach that searches through the metagenomic "trash bin" for genes associated with conjugative systems present on contigs that are typically excluded from common metagenomic analysis pipelines.

A generalizable Cas9/sgRNA prediction model using machine transfer learning with small high-quality datasets.

The CRISPR/Cas9 nuclease from Streptococcus pyogenes (SpCas9) can be used with single guide RNAs (sgRNAs) as a sequence-specific antimicrobial agent and as a genome-engineering tool. However, current bacterial sgRNA activity models struggle with accurate predictions and do not generalize well, possibly because the underlying datasets used to train the models do not accurately measure SpCas9/sgRNA activity and cannot distinguish on-target cleavage from toxicity. Here, we solve this problem by using a two-plasmid positive selection system to generate high-quality data that more accurately reports on SpCas9/sgRNA cleavage and that separates activity from toxicity.

Noncanonical functions of Ku may underlie essentiality in human cells.

The Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair but is involved in other cellular functions like telomere regulation and maintenance, in which Ku's role is not fully characterized. It was previously reported that knockout of Ku80 in a human cell line results in lethality, but the underlying cause of Ku essentiality in human cells has yet to be fully explored. Here, we established conditional Ku70 knockout cells using CRISPR/Cas9 editing to study the essentiality of Ku70 function.

Metabolically-targeted dCas9 expression in bacteria.

The ability to restrict gene expression to a relevant bacterial species in a complex microbiome is an unsolved problem. In the context of the human microbiome, one desirable target metabolic activity are glucuronide-utilization enzymes (GUS) that are implicated in the toxic re-activation of glucuronidated compounds in the human gastrointestinal (GI) tract, including the chemotherapeutic drug irinotecan. Here, we take advantage of the variable distribution of GUS enzymes in bacteria as a means to distinguish between bacteria with GUS activity, and re-purpose the glucuronide-responsive GusR transcription factor as a biosensor to regulate dCas9 expression in response to glucuronide inducers.

Superior Conjugative Plasmids Delivered by Bacteria to Diverse Fungi.

Fungi are nature's recyclers, allowing for ecological nutrient cycling and, in turn, the continuation of life on Earth. Some fungi inhabit the human microbiome where they can provide health benefits, while others are opportunistic pathogens that can cause disease. Yeasts, members of the fungal kingdom, have been domesticated by humans for the production of beer, bread, and, recently, medicine and chemicals.

Telomere-to-telomere genome assembly of .

is a marine diatom with a growing genetic toolbox available and is being used in many synthetic biology applications. While most of the genome has been assembled, the currently available genome assembly is not a completed telomere-to-telomere assembly. Here, we used Oxford Nanopore long reads to build a telomere-to-telomere genome for .

Conjugation-Based Genome Engineering in .

has become an attractive microbial platform for the study of extremophile biology and industrial bioproduction. To improve the genomic manipulation and tractability of this species, the development of tools for whole genome engineering and design is necessary. Here, we report the development of a simple and robust conjugation-based DNA transfer method from to , allowing for the introduction of stable, replicating plasmids expressing antibiotic resistance markers.

Cloning of Mitochondrial Genome in and .

Algae are attractive organisms for biotechnology applications such as the production of biofuels, medicines, and other high-value compounds due to their genetic diversity, varied physical characteristics, and metabolic processes. As new species are being domesticated, rapid nuclear and organelle genome engineering methods need to be developed or optimized. To that end, we have previously demonstrated that the mitochondrial genome of microalgae can be cloned and engineered in and .

Plasmid-based complementation of large deletions in Phaeodactylum tricornutum biosynthetic genes generated by Cas9 editing.

The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes.

Trans-Kingdom Conjugation within Solid Media from to .

Conjugation is a bacterial mechanism for DNA transfer from a donor cell to a wide range of recipients, including both prokaryotic and eukaryotic cells. In contrast to conventional DNA delivery techniques, such as electroporation and chemical transformation, conjugation eliminates the need for DNA extraction, thereby preventing DNA damage during isolation. While most established conjugation protocols allow for DNA transfer in liquid media or on a solid surface, we developed a procedure for conjugation within solid media.

Modifying a covarying protein-DNA interaction changes substrate preference of a site-specific endonuclease.

Identifying and validating intermolecular covariation between proteins and their DNA-binding sites can provide insights into mechanisms that regulate selectivity and starting points for engineering new specificity. LAGLIDADG homing endonucleases (meganucleases) can be engineered to bind non-native target sites for gene-editing applications, but not all redesigns successfully reprogram specificity. To gain a global overview of residues that influence meganuclease specificity, we used information theory to identify protein-DNA covariation.

Efficient inter-species conjugative transfer of a CRISPR nuclease for targeted bacterial killing.

The selective regulation of bacteria in complex microbial populations is key to controlling pathogenic bacteria. CRISPR nucleases can be programmed to kill bacteria, but require an efficient and broad-host range delivery system to be effective. Here, using an Escherichia coli and Salmonella enterica co-culture system, we show that plasmids based on the IncP RK2 conjugative system can be used as delivery vectors for a TevSpCas9 dual nuclease.

Designer Sinorhizobium meliloti strains and multi-functional vectors enable direct inter-kingdom DNA transfer.

Storage, manipulation and delivery of DNA fragments is crucial for synthetic biology applications, subsequently allowing organisms of interest to be engineered with genes or pathways to produce desirable phenotypes such as disease or drought resistance in plants, or for synthesis of a specific chemical product. However, DNA with high G+C content can be unstable in many host organisms including Saccharomyces cerevisiae. Here, we report the development of Sinorhizobium meliloti, a nitrogen-fixing plant symbioticα-Proteobacterium, as a novel host that can store DNA, and mobilize DNA to E.

Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases.

LAGLIDADG homing endonucleases (meganucleases) are site-specific mobile endonucleases that can be adapted for genome-editing applications. However, one problem when reprogramming meganucleases on non-native substrates is indirect readout of DNA shape and flexibility at the central 4 bases where cleavage occurs. To understand how the meganuclease active site regulates DNA cleavage, we used functional selections and deep sequencing to profile the fitness landscape of 1600 I-LtrI and I-OnuI active site variants individually challenged with 67 substrates with central 4 base substitutions.

Delivery of the Cas9 or TevCas9 system into via conjugation of plasmids from a bacterial donor.

Diatoms are an ecologically important group of eukaryotic microalgae with properties that make them attractive for biotechnological applications such as biofuels, foods, cosmetics and pharmaceuticals. is a model diatom with defined culture conditions, but routine genetic manipulations are hindered by a lack of simple and robust genetic tools. One obstacle to efficient engineering of is that the current selection methods for transformants depend on the use of a limited number of antibiotic resistance genes.

mitoTev-TALE: a monomeric DNA editing enzyme to reduce mutant mitochondrial DNA levels.

Pathogenic mitochondrial DNA (mtDNA) mutations often co-exist with wild-type molecules (mtDNA heteroplasmy). Phenotypes manifest when the percentage of mutant mtDNA is high (70-90%). Previously, our laboratory showed that mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs) can eliminate mutant mtDNA from heteroplasmic cells.

Repression of branched-chain amino acid synthesis in Staphylococcus aureus is mediated by isoleucine via CodY, and by a leucine-rich attenuator peptide.

Staphylococcus aureus requires branched-chain amino acids (BCAAs; isoleucine, leucine, valine) for protein synthesis, branched-chain fatty acid synthesis, and environmental adaptation by responding to their availability via the global transcriptional regulator CodY. The importance of BCAAs for S. aureus physiology necessitates that it either synthesize them or scavenge them from the environment.

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum.

With the completion of the genome sequence, and development of an efficient conjugation-based transformation system allowing the introduction of stable episomes, Phaeodactylum tricornutum has become an ideal platform for the study of diatom biology and synthetic biology applications. The development of plasmid-based genetic tools is the next step to improve manipulation of this species. Here, we report the identification of endogenous P.

Applications of Alternative Nucleases in the Age of CRISPR/Cas9.

Breakthroughs in the development of programmable site-specific nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases (MNs), and most recently, the clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins (including Cas9) have greatly enabled and accelerated genome editing. By targeting double-strand breaks to user-defined locations, the rates of DNA repair events are greatly enhanced relative to un-catalyzed events at the same sites. However, the underlying biology of each genome-editing nuclease influences the targeting potential, the spectrum of off-target cleavages, the ease-of-use, and the types of recombination events at targeted double-strand breaks.