Multiplexed activation in mammalian cells using a split-intein CRISPR/Cas12a based synthetic transcription factor

The adoption of CRISPR systems for the generation of synthetic transcription factors has greatly simplified the process for upregulating endogenous gene expression, with a plethora of applications in cell biology, bioproduction and cell reprogramming. The recently discovered CRISPR/Cas12a (Cas12a) systems offer extended potential, as Cas12a is capable of processing its own crRNA array, to provide multiple individual crRNAs for subsequent targeting from a single transcript. Here we show the application of dFnCas12a-VPR in mammalian cells, with the Francisella novicida Cas12a (FnCas12a) possessing a shorter PAM sequence than Acidaminococcus sp. (As) or Lachnospiraceae bacterium (Lb) variants, enabling denser targeting of genomic loci, while performing just as well or even better than the other variants. We observe that synergistic activation and multiplexing can be achieved using crRNA arrays but also show that crRNAs expressed towards the 5′ of 6-crRNA arrays show evidence of enhanced activity. This not only represents a more flexible tool for transcriptional modulation but further expands our understanding of the design capabilities and limitations when considering longer crRNA arrays for multiplexed targeting

Standardization of synthetic biology tools and assembly methods for Saccharomyces cerevisiae and emerging yeast species

As redesigning organisms using engineering principles is one of the purposes of synthetic biology (SynBio), the standardization of experimental methods and DNA parts is becoming increasingly a necessity. The synthetic biology community focusing on the engineering of Saccharomyces cerevisiae has been in the foreground in this area, conceiving several well-characterized SynBio toolkits widely adopted by the community. In this review, the molecular methods and toolkits developed for S. cerevisiae are discussed in terms of their contributions to the required standardization efforts. In addition, the toolkits designed for emerging nonconventional yeast species including Yarrowia lipolytica, Komagataella phaffii, and Kluyveromyces marxianus are also reviewed. Without a doubt, the characterized DNA parts combined with the standardized assembly strategies highlighted in these toolkits have greatly contributed to the rapid development of many metabolic engineering and diagnostics applications among others. Despite the growing capacity in deploying synthetic biology for common yeast genome engineering works, the yeast community has a long journey to go to exploit it in more sophisticated and delicate applications like bioautomation.ISSN:2161-506

YeastFab:the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae

It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for production of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biological parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biological parts, which can be used to assemble transcriptional units in a single-tube reaction. In addition, standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using β-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the production through combinatorial assembly of a pathway using hundreds of regulatory biological parts

Establishing a standardised DNA assembly and biosensor-based screening pipeline for natural product pathway engineering in Saccharomyces cerevisiae

Many pharmaceutical drugs currently available on the market are natural products, which are extracted from biological sources (microorganisms or plants). It can prove challenging to maintain a constant supply of these drugs, considering that they are often derived from microorganisms that are difficult to culture or plants, where availability can be strongly impacted by demand for alternative crops or severe weather conditions. Synthetic biology presents a promising and more sustainable option, through the engineering of easily cultured and genetically tractable microorganisms such as Saccharomyces cerevisiae to enable their production of pharmaceutical compounds. However, the process of heterologous expression of a bacteria-derived natural product pathway in S. cerevisiae can be challenging. Many variables need to be adjusted, such as codon optimisation, gene expression, pathway flux, enzyme variants utilised and the activity of different enzymes within the pathway. In the process of optimisation and screening, a major rate-limiting step is the assembly of diverse pathway variants, which can be time consuming and costly. The aim of this project was to utilise synthetic biology tools and techniques to engineer yeast strains to produce different therapeutic compounds. This involves the establishment of a pipeline to be able to 1) assemble many different variants of large pathways, and 2) rapidly screen for the production of a bioactive compound and identify producer or high producer strains. In this body of work, I describe the development of YeastFab, a toolkit of standardised and modular biological yeast ‘parts’ that can be used to hierarchically assemble large natural product pathways that encode for production of therapeutic drugs within the span of a week. To date, hundreds of promoters and terminators have been standardised into the YeastFab format and have been further characterised to determine their activities. This assembly strategy is subsequently applied in the context of a collaborative project, funded by the Bill and Melinda Gates Foundation, for heterologous expression of exemplar pathways from different classes of natural products (NRPS, PKS, RiPPs, nucleosides, alkaloids and flavonoids). I have primarily focussed on the heterologous production of viomycin, rebeccamycin and naringenin. Different variants of each pathway have been assembled and screened using a bioassay to detect antibiotic production or using a biosensor specific to the compound of interest. As a proof of principle, I have developed a high throughput assay, utilising an Escherichia coli transcription factor-based biosensor to quantify the amount of naringenin produced by each yeast strain, demonstrating comparable quantification to mass spectrometry measurements. Further improvements of naringenin yield are being carried out through chassis optimisation by genome engineering techniques such as SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxPsyn-mediated Evolution), with the use of the E. coli biosensor to sift through the population of SCRaMbLEd strains for high producers. In conclusion, I have developed and demonstrated a pipeline for rapid assembly and screening of natural products biosynthetic pathways in yeast, which can facilitate the development of novel processes for manufacture of many therapeutic compounds

EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors

Mammalian plasmid expression vectors are critical reagents underpinning many facets of research across biology, biomedical research, and the biotechnology industry. Traditional cloning methods often require laborious manual design and assembly of plasmids using tailored sequential cloning steps. This process can be protracted, complicated, expensive, and error-prone. New tools and strategies that facilitate the efficient design and production of bespoke vectors would help relieve a current bottleneck for researchers. To address this, we have developed an extensible mammalian modular assembly kit (EMMA). This enables rapid and efficient modular assembly of mammalian expression vectors in a one-tube, one-step golden-gate cloning reaction, using a standardized library of compatible genetic parts. The high modularity, flexibility, and extensibility of EMMA provide a simple method for the production of functionally diverse mammalian expression vectors. We demonstrate the value of this toolkit by constructing and validating a range of representative vectors, such as transient and stable expression vectors (transposon based vectors), targeting vectors, inducible systems, polycistronic expression cassettes, fusion proteins, and fluorescent reporters. The method also supports simple assembly combinatorial libraries and hierarchical assembly for production of larger multigenetic cargos. In summary, EMMA is compatible with automated production, and novel genetic parts can be easily incorporated, providing new opportunities for mammalian synthetic biology