The genetic engineering of genome fragments larger than 100 kbp is challenging and requires both specific methods and cloning hosts. The yeast Saccharomyces cerevisiae is considered as a host of choice for cloning and engineering whole or partial genomes from viruses, bacteria, and algae. Several methods are now available to perform these manipulations, each with its own limitations. In order to extend the range of yeast cloning strategies, a new approach combining two already described methods, Fusion cloning and CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by the fusion of bacterial cells with yeast spheroplasts carrying the CRISPR-Cas9 system. With this new approach, we demonstrate the feasibility of cloning and editing whole genomes from several Mycoplasma species belonging to different phylogenetic groups. We also show that CReasPy-Fusion allows the capture of large genome fragments with high efficacy, resulting in the successful cloning of selected loci in yeast. We finally identify bacterial nuclease encoding genes as barriers for CReasPy-Fusion by showing that their removal from the donor genome improves the cloning efficacy.

The ability to finely tune reaction rates and binding energies between components has made DNA strand displacement circuits promising candidates to replicate the complex regulatory functions of biological reaction networks. However, these circuits often lack crucial properties, such as signal turnover and the ability to transiently respond to successive input signals that require the continuous input of chemical energy. Here, we introduce a method for providing such energy to strand displacement networks in a controlled fashion: an engineered DNA helicase, Rep-X, that transiently dehybridizes specific DNA complexes, enabling the strands in the complex to participate in downstream hybridization or strand displacement reactions. We demonstrate how this process can direct the formation of specific metastable structures by design and that this dehybridization process can be controlled by DNA strand displacement reactions that effectively protect and deprotect a double-stranded complex from unwinding by Rep-X. These findings can guide the design of active DNA strand displacement regulatory networks, in which sustained dynamical behavior is fueled by helicase-regulated unwinding.

Staphylococcus aureus is a clinically important pathogen that threatens human health due to its strong pathogenicity and drug resistance, leading to meningitis, endocarditis, and skin and soft tissue infections. Genetic manipulation in S.aureus is a powerful approach for characterizing the molecular mechanisms of bacterial drug resistance, pathogenicity, and virulence. However, a strong restriction barrier presents a major obstacle to the extensive utilization of genetic manipulation tools in clinical isolates of S.aureus. Here, we constructed a restriction-modification (RM) system silent CRISPR-Cas9 toolkit that synonymously eliminated the type I RM targets of S.aureus from plasmids, downsized plasmids using minicircle technology, and combined with a plasmid artificial modification (PAM) method to circumvent the type II RM system. The RM-silent CRISPR-Cas9 toolkit enables a significant improvement in transformation (105-106 transformants per microgram plasmid in strains we tested) and high-success efficiency editing for gene deletion (knockout strain obtained in one-round electroporation) in a wide range of S.aureus species including clinical isolates of unknown genetic background. The RM-silent CRISPR-Cas9 toolkits could expedite the process of mutant construction in most S.aureus strains, and this approach could be applied to the design of other genetic toolkit plasmids for utilization in a wider range of S.aureus strains.

Advanced microbiome therapeutics (AMTs) holds promise in utilizing engineered microbes such as bacteria or yeasts for innovative therapeutic applications, including the in situ delivery of therapeutic peptides. Glucagon-like peptide-1 receptor agonists, such as Exendin-4, have emerged as potential treatments for type 2 diabetes and obesity. However, current administration methods face challenges with patient adherence and low oral bioavailability. To address these limitations, researchers are exploring improved oral delivery methods for Exendin-4, including utilizing AMTs. This study engineered the probiotic yeast Saccharomyces boulardii to produce Exendin-4 (Sb-Exe4) in the gastrointestinal tract of male C57BL/6 mice to combat diet-induced obesity. The biological efficiency of Exendin-4 secreted by S. boulardii was analyzed ex vivo on isolated pancreatic islets, demonstrating induced insulin secretion. The in vivo characterization of Sb-Exe4 revealed that when combined with cold exposure (8 °C), the Sb-Exe4 yeast strain successfully suppressed appetite by 25% and promoted a 4-fold higher weight loss. This proof of concept highlights the potential of AMTs to genetically modify S. boulardii for delivering active therapeutic peptides in a precise and targeted manner. Although challenges in efficacy and regulatory approval persist, AMTs may provide a transformative platform for personalized medicine. Further research in AMTs, particularly focusing on probiotic yeasts such as S. boulardii, holds great potential for novel therapeutic possibilities and enhancing treatment outcomes in diverse metabolic disorders.

In this study, a suite of efficient CRISPR/Cas9 tools was developed to overcome the genetic manipulation challenges posed by the polyploid genome of industrial yeast Cyberlindnera jadinii. The developed CRISPR/Cas9 system can achieve a 100% single-gene knockdown efficiency in strain NBRC0988. Moreover, the integration of a single exogenous gene into the target locus using a 50 bp homology arm achieved near-100% efficiency. The efficiency of simultaneous integration of three genes into the chromosome is strongly influenced by the length of the homology arm, with the highest integration efficiency of 62.5% obtained when selecting a homology arm of about 500 bp. By utilizing the CRISPR/Cas system, this study demonstrated the potential of C. jadinii in producing heterologous sterols. Through shake-flask fermentation, the engineered strains produced 92.1 and 81.8 mg/L of campesterol and cholesterol, respectively. Furthermore, the production levels of these two sterols were further enhanced through high-cell-density fed-batch fermentation in a 5 L bioreactor. The highest titer of campesterol reached 807 mg/L [biomass OD600 = 294, productivity of 6.73 mg/(L·h)]. The titer of cholesterol reached 1.52 g/L [biomass OD600 = 380, productivity of 9.06 mg/(L·h)], marking the first gram-scale production of steroidal compounds in C. jadinii.

Gene expression control based on clustered regularly interspaced short palindromic repeats (CRISPR) has emerged as a powerful approach for constructing synthetic gene circuits. While the use of CRISPR interference (CRISPRi) is already well-established in prokaryotic circuits, CRISPR activation (CRISPRa) is less mature, and a combination of the two in the same circuits is only just emerging. Here, we report that combining CRISPRi with SoxS-based CRISPRa in Escherichia coli can lead to context-dependent effects due to different affinities in the formation of CRISPRa and CRISPRi complexes, resulting in loss of predictable behavior. We show that this effect can be avoided by using the same scaffold guide RNA structure for both complexes.

Microorganisms (mainly bacteria and yeast) are frequently used as hosts for genetic constructs in synthetic biology applications. Molecular noise might have a significant effect on the dynamics of gene regulation in microbial cells, mainly attributed to the low copy numbers of mRNA species involved. However, the inclusion of molecular noise in the automated design of biocircuits is not a common practice due to the computational burden linked to the chemical master equation describing the dynamics of stochastic gene regulatory circuits. Here, we address the automated design of synthetic gene circuits under the effect of molecular noise combining a mixed integer nonlinear global optimization method with a partial integro-differential equation model describing the evolution of stochastic gene regulatory systems that approximates very efficiently the chemical master equation. We demonstrate the performance of the proposed methodology through a number of examples of relevance in synthetic biology, including different bimodal stochastic gene switches, robust stochastic oscillators, and circuits capable of achieving biochemical adaptation under noise.

Streptomyces, a genus of Gram-positive bacteria, is known as nature's medicine maker, producing a plethora of natural products that have huge benefits for human health, agriculture, and biotechnology. To take full advantage of this treasure trove of bioactive molecules, better genetic tools are required for the genetic engineering and synthetic biology of Streptomyces. We therefore developed CUBIC, a novel CUmate-Based Inducible CRISPR interference (CRISPRi) system that allows highly efficient and inducible gene knockdown in Streptomyces. Its broad application is shown by the specific and nondisruptive knockdown of genes involved in growth, development and antibiotic production in various Streptomyces species. To facilitate hyper-efficient plasmid construction, we adapted the Golden Gate assembly to achieve 100% cloning efficiency of the protospacers. We expect that the versatile plug-and-play CUBIC system will create new opportunities for research and innovation in the field of Streptomyces.

As living drugs, engineered T cell therapies are revolutionizing disease treatment with their unique functional capabilities. However, they suffer from limitations of potentially unpredictable behavior, toxicities, and nontraditional pharmacokinetics. Engineering conditional control mechanisms responsive to tractable stimuli such as small molecules or light is thus highly desirable. We and others previously developed "universal" chimeric antigen receptors (CARs) that interact with coadministered antibody adaptors to direct target cell killing and T cell activation. Universal CARs are of high therapeutic interest due to their ability to simultaneously target multiple antigens on the same disease or different diseases by combining with adaptors to different antigens. Here, we further enhance the programmability and potential safety of universal CAR T cells by engineering OFF-switch adaptors that can conditionally control CAR activity, including T cell activation, target cell lysis, and transgene expression, in response to a small molecule or light stimulus. Moreover, in adaptor combination assays, OFF-switch adaptors were capable of orthogonal conditional targeting of multiple antigens simultaneously, following Boolean logic. OFF-switch adaptors represent a robust new approach for the precision targeting of universal CAR T cells with potential for enhanced safety.

Splitting proteins with light- or chemically inducible dimers provides a mechanism for post-translational control of protein function. However, current methods for engineering stimulus-responsive split proteins often require significant protein engineering expertise and the laborious screening of individual constructs. To address this challenge, we use a pooled library approach that enables rapid generation and screening of nearly all possible split protein constructs in parallel, where results can be read out by using sequencing. We perform our method on Cre recombinase with optogenetic dimers as a proof of concept, resulting in comprehensive data on the split sites throughout the protein. To improve the accuracy in predicting split protein behavior, we develop a Bayesian computational approach to contextualize errors inherent to experimental procedures. Overall, our method provides a streamlined approach for achieving inducible post-translational control of a protein of interest.