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Conventional CRISPR approaches for precision genome editing rely on the introduction of DNA double-strand breaks (DSB) and activation of homology-directed repair (HDR), which is inherently genotoxic and inefficient in somatic cells. The development of base editing (BE) systems that edit a target base without requiring generation of DSB or HDR offers an alternative. Here, we describe a novel BE system called Pin-point(TM) that recruits a DNA base-modifying enzyme through an RNA aptamer within the gRNA molecule. Pin-point is capable of efficiently modifying base pairs in the human genome with precision and low on-target indel formation. This system can potentially be applied for correcting pathogenic mutations, installing premature stop codons in pathological genes, and introducing other types of genetic changes for basic research and therapeutic development.

Engineered immune cell therapy is revolutionising the field of cancer therapeutics. US Food and Drug Administration (FDA) approval of two chimeric antigen receptor (CAR)-T cell products for the treatment of haematological malignancies paved the way for individualised cancer treatment. However, multiple genetic edits will be required to improve the efficacy of CAR-T cell therapies if they are to treat refractory malignancies successfully, particularly solid tumours. Off-target effects of CRISPR–Cas9-mediated multiplex editing are likely to hinder its safety and application in the clinic. Novel base editing technologies offer a promising and safer alternative for simultaneous editing that could enhance allogeneic engineered immunotherapies for targeting solid tumours and other complex human diseases.

Improved health span and lifespan extension in a wide phylogenetic range of species is associated with the induction of the environmental cell stress response through a signalling pathway regulated by the transcription factor Nrf2. Phytochemicals which stimulate this response may form part of therapeutic interventions which stimulate endogenous cytoprotective mechanisms, thereby delaying the onset of age-related diseases and promoting healthy ageing in humans. In order to identify compounds that activate the Nrf2 pathway, a cell-based reporter system was established in HepG2 cells using a luciferase reporter gene under the control of the Nqo1 promoter. Sulforaphane, an isothiocyanate derived from cruciferous vegetables and a known activator of the Nrf2 pathway, was used to validate the reporter system. The transfected cell line HepG2 C1 was subsequently used to screen natural product libraries. Five compounds were identified as activating the bioluminescent reporter by greater than 5-fold. The two most potent compounds, MBC20 and MBC37, were further characterised and shown to stimulate endogenous cytoprotective gene and protein expression. The bioluminescent reporter system will allow rapid, in vitro identification of novel compounds that have the potential to improve health span through activation of the environmental stress response.

Site-specific recombinases are important tools for the modification of mammalian genomes. In conjunction with viral vectors, they can be utilized to mediate site-specific gene insertions in animals and in cell lines which are difficult to transfect. Here we describe a method for the generation and analysis of an adenovirus vector supporting a recombinase-mediated cassette exchange reaction and discuss the advantages and limitations of this approach.

Site-specific recombinases are important tools for the modification of mammalian genomes. In conjunction with viral vectors, they can be utilized to mediate site-specific gene insertions in animals and in cell lines which are difficult to transfect. Here we describe a method for the generation and analysis of an adenovirus vector supporting a recombinase-mediated cassette exchange reaction and discuss the advantages and limitations of this approach.

Improved health span and lifespan extension in a wide phylogenetic range of species is associated with upregulation of the environmental stress response (ESR). The ESR, driven by the transcription factor Nrf2, provides endogenous antioxidant defence to maintain intracellular redox balance. Phytochemicals which stimulate the ESR thus upregulate cytoprotective mechanisms and ultimately contribute towards delaying the onset of age-related degenerative diseases. Two bioluminescent reporter cell lines were constructed to identify and characterise inducers of the ESR, specifically induction of Nrf2 target genes NQO1 and HMOX1 . The first cell line was generated using random integration of an exogenous luciferase gene under control of an NQO1 gene promoter, and allowed rapid and sensitive high throughput screening of natural chemical libraries. The second assay system employed a more targeted strategy using an adeno-associated virus (AAV) vector to mediate the site-specific integration of a luciferase reporter into the locus of the endogenous ESR gene HMOX1 , thus allowing predictive indexing of the gene’s transcription. The two cell lines were validated with potent ESR inducers sulforaphane and curcumin. A compound identified in the screening of a chemical library from the Marine Biodiscovery Centre (University of Aberdeen, UK) was further characterised in a microarray experiment and compared to known phytochemicals. In combination, these in vitro cell-based assay systems will allow a better understanding of the activity-structure relationship of ESR inducers, and assess the bioactivity of phytochemical metabolites from human serum samples following intervention trials.