CRISPR-based self-cleaving mechanism for controllable gene delivery in human cells

Controllable gene delivery via vector-based systems remains a formidable challenge in mammalian synthetic biology and a desirable asset in gene therapy applications. Here, we introduce a methodology to control the copies and residence time of a gene product delivered in host human cells but also selectively disrupt fragments of the delivery vehicle. A crucial element of the proposed system is the CRISPR protein Cas9. Upon delivery, Cas9 guided by a custom RNA sequence cleaves the delivery vector at strategically placed targets thereby inactivating a co-expressed gene of interest. Importantly, using experiments in human embryonic kidney cells, we show that specific parameters of the system can be adjusted to fine-tune the delivery properties. We envision future applications in complex synthetic biology architectures, gene therapy and trace-free delivery.     

DNA profiling

Although some concerns still remain in standard DNA profiling technology over the assumptions from population genetics used to calculate expected match frequencies, forensic scientists are preparing for the introduction of the next generation of DNA profiling techniques based on the polymerase chain reaction. These new techniques offer the prospect of dramatically increasing the speed and sensitivity of DNA profiling and have already been applied in some casework studies.     

Sensitive detection of glucagon aggregation using amyloid fibril-specific antibodies

Sensitive detection of protein aggregates is important for evaluating the quality of biopharmaceuticals and detecting misfolded proteins in several neurodegenerative diseases. However, it is challenging to detect extremely low concentrations (<10 ppm) of aggregated protein in the presence of high concentrations of soluble protein. Glucagon, a peptide hormone used in the treatment of extreme hypoglycemia, is aggregation-prone and forms amyloid fibrils. Detection of glucagon fibrils using conformation-specific antibodies is an attractive approach for identifying such aggregates during process and formulation development. Therefore, we have used yeast surface display and magnetic-activated cell sorting to sort single-chain antibody libraries to identify antibody variants with high conformational specificity for glucagon fibrils. Notably, we find several high-affinity antibodies that display excellent selectivity for glucagon fibrils, and we have integrated these antibodies into a sensitive immunoassay. Surprisingly, the sensitivity of our assay—which involves direct (nonantibody mediated) glucagon immobilization in microtiter plates—can be significantly enhanced by pretreating the microtiter plates with various types of globular proteins before glucagon immobilization. Moreover, increased total concentrations of glucagon peptide also significantly improve the sensitivity of our assay, which appears to be due to the strong seeding activity of immobilized fibrils at high glucagon concentrations. Our final assay is highly sensitive (fibril detection limit of ~0.5–1 ppm) and is >20 times more sensitive than detection using a conventional, amyloid-specific fluorescent dye (Thioflavin T). We expect that this type of sensitive immunoassay can be readily integrated into the drug development process to improve the generation of safe and potent peptide therapeutics.     

Functional Genomics Identifies Metabolic Vulnerabilities in Pancreatic Cancer

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Transmission Distortion Affecting Human Noncrossover but Not Crossover Recombination: A Hidden Source of Meiotic Drive

Meiotic recombination ensures the correct segregation of homologous chromosomes during gamete formation and contributes to DNA diversity through both large-scale reciprocal crossovers and very localised gene conversion events, also known as noncrossovers. Considerable progress has been made in understanding factors such as PRDM9 and SNP variants that influence the initiation of recombination at human hotspots but very little is known about factors acting downstream. To address this, we simultaneously analysed both types of recombinant molecule in sperm DNA at six highly active hotspots, and looked for disparity in the transmission of allelic variants indicative of any cis-acting influences. At two of the hotspots we identified a novel form of biased transmission that was exclusive to the noncrossover class of recombinant, and which presumably arises through differences between crossovers and noncrossovers in heteroduplex formation and biased mismatch repair. This form of biased gene conversion is not predicted to influence hotspot activity as previously noted for SNPs that affect recombination initiation, but does constitute a powerful and previously undetected source of recombination-driven meiotic drive that by extrapolation may affect thousands of recombination hotspots throughout the human genome. Intriguingly, at both of the hotspots described here, this drive favours strong (G/C) over weak (A/T) base pairs as might be predicted from the well-established correlations between high GC content and recombination activity in mammalian genomes.     

The role of pre-release efficacy assessment in selecting classical biological control agents for weeds—applying the Anna Karenina principle

The goals in selecting classical biological control agents for weeds are to identify agents that will be both safe for release and effective in controlling their target plants. The release of ineffective agents should be avoided, as these add to the costs and risks of biological control without contributing to its benefits. While the principles of host-specificity testing and risk assessment for weed biological control agents have been extensively debated and refined, there has been less attention given to assessing the probable efficacy of agents prior to release. This reluctance to undertake pre-release efficacy assessment (PREA) is probably based on concerns that it will both add to the cost of screening biological control agents and introduce a risk of wrongly rejecting effective agents. We used a project simulation model to investigate the implications of using PREA as an additional filter in the agent selection process. The results suggest that, if it can be done at a lower cost than host-specificity testing, the use of PREA as the first filter can make agent selection more cost-effective than screening based on host-specificity alone. We discuss examples of PREA and potential approaches. The impact of biocontrol agents is a function of their range, abundance, and per-capita damage. While it will always be difficult to predict the post-release abundance of biological control agents from pre-release studies, some estimates of potential range can be obtained from studies of climatic adaptation. For agents that affect the vegetative growth or survival of their target weeds, experimental measurement of per-capita damage is feasible and can contribute to a reduction in the numbers of ineffective agents released. The Anna Karenina principle states that success in complex undertakings does not depend on a single factor but requires avoiding many separate causes of failure. We suggest that, in biological control of weeds, the use of agents that are not sufficiently damaging is one such cause that can be partially avoided by the use of pre-release efficacy assessment.