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CRISPR-based diagnostics enable specific sensing of DNA and RNA biomarkers associated with human diseases. This is achieved through the binding of guide RNAs to a complementary sequence that activates Cas enzymes to cleave reporter molecules. Currently, most CRISPR-based diagnostics rely on target preamplification to reach sufficient sensitivity for clinical applications. This limits quantification capability and adds complexity to the reaction chemistry. Here we show the combination of a CRISPR–Cas-based reaction with a nanozyme-linked immunosorbent assay, which allows for the quantitative and colorimetric readout of Cas13-mediated RNA detection through catalytic metallic nanoparticles at room temperature (CrisprZyme). We demonstrate that CrisprZyme is easily adaptable to a lateral-flow-based readout and different Cas enzymes and enables the sensing of non-coding RNAs including microRNAs, long non-coding RNAs and circular RNAs. We utilize this platform to identify patients with acute myocardial infarction and to monitor cellular differentiation in vitro and in tissue biopsies from prostate cancer patients. We anticipate that CrisprZyme will serve as a universally applicable signal catalyst for CRISPR-based diagnostics, which will expand the spectrum of targets for preamplification-free, quantitative detection.The combination of catalytic platinum particles, nanozymes and a CRISPR-based reaction allows for the quantification of non-coding RNAs at the picomolar range. This assay, CrisprZyme, has a colorimetric readout and works at room temperature without preamplification.

In organ transplantation, infection and rejection are major causes of graft loss. They are linked by the net state of immunosuppression. To diagnose and treat these conditions earlier, and to improve long-term patient outcomes, refined strategies for the monitoring of patients after graft transplantation are needed. Here, we show that a fast and inexpensive assay based on CRISPR–Cas13 accurately detects BK polyomavirus DNA and cytomegalovirus DNA from patient-derived blood and urine samples, as well as CXCL9 messenger RNA (a marker of graft rejection) at elevated levels in urine samples from patients experiencing acute kidney transplant rejection. The assay, which we adapted for lateral-flow readout, enables—via simple visualization—the post-transplantation monitoring of common opportunistic viral infections and of graft rejection, and should facilitate point-of-care post-transplantation monitoring. A fast and inexpensive point-of-care assay based on CRISPR–Cas13 accurately detects the DNA of opportunistic viruses in blood and urine samples as well as an mRNA marker of renal transplant rejection in urine samples.

Non-viral gene editing via CRISPR-Cas12a offers an alternative to Cas9-based methods, providing better targeting of AT-rich regions, simplified guide RNA manufacturing, and high specificity. However, the efficacy of editing outcomes is subject to various factors, with template format playing a crucial role. Currently, the predominant non-viral template format for inducing homology-directed repair (HDR) after nuclease-induced DNA breaks is double-stranded DNA (dsDNA), which is toxic when transfected at high doses. Previous studies have demonstrated that using single-stranded DNA (ssDNA) with flanking double-stranded Cas-target-sequences (CTS) as a repair template for Cas9-mediated gene editing can mitigate this toxicity and increase knock-in efficiency. Here, we investigate CTS design for AsCas12a Ultra by exploring PAM orientation and binding requirements of the Cas12a-crRNA complex. Additionally, we rule out in vitro ssDNase activity of AsCas12a Ultra under cell-physiological Mg2+ conditions. Finally, we showcase the advantage of using ssDNA with double-stranded CTS end modifications (ssCTS) at high doses for delivering clinically relevant transgenes of varying sizes into three T-cell receptor-CD3 complex genes (TRAC, CD3ζ, CD3ε), achieving up to 90% knock-in rates for a 0.8kb insert at the CD3ε locus. Overall, AsCas12a Ultra and ssCTS donors represent a platform for highly efficient knock-in in primary human T cells with minimal toxicity.

Dipeptide repeat proteins are a major pathogenic feature of C9orf72 amyotrophic lateral sclerosis (C9ALS)/frontotemporal dementia (FTD) pathology, but their physiological impact has yet to be fully determined. Here we generated C9orf72 dipeptide repeat knock-in mouse models characterized by expression of 400 codon-optimized polyGR or polyPR repeats, and heterozygous C9orf72 reduction. (GR)400 and (PR)400 knock-in mice recapitulate key features of C9ALS/FTD, including cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. TGF-β1 was one of the top predicted regulators of this ECM signature and polyGR expression in human induced pluripotent stem cell neurons was sufficient to induce TGF-β1 followed by COL6A1. Knockdown of TGF-β1 or COL6A1 orthologues in polyGR model Drosophila exacerbated neurodegeneration, while expression of TGF-β1 or COL6A1 in induced pluripotent stem cell-derived motor neurons of patients with C9ALS/FTD protected against glutamate-induced cell death. Altogether, our findings reveal a neuroprotective and conserved ECM signature in C9ALS/FTD. C9orf72 ALS/FTD polyGR and polyPR knock-in mice show cortical hyperexcitability and motor neuron loss accompanied by an increase in extracellular matrix proteins in the spinal cord that is conserved in patient iPS cell-derived neurons and is neuroprotective.

Mutations in the enzyme glycyl-tRNA synthetase (GARS) cause motor and sensory axon loss in the peripheral nervous system in humans, described clinically as Charcot-Marie-Tooth type 2D or distal spinal muscular atrophy type V. Here, we characterise a new mouse mutant, GarsC201R, with a point mutation that leads to a non-conservative substitution within GARS. Heterozygous mice with a C3H genetic background have loss of grip strength, decreased motor flexibility and disruption of fine motor control; this relatively mild phenotype is more severe on a C57BL/6 background. Homozygous mutants have a highly deleterious set of features, including movement difficulties and death before weaning. Heterozygous animals have a reduction in axon diameter in peripheral nerves, slowing of nerve conduction and an alteration in the recovery cycle of myelinated axons, as well as innervation defects. An assessment of GARS levels showed increased protein in 15-day-old mice compared with controls; however, this increase was not observed in 3-month-old animals, indicating that GARS function may be more crucial in younger animals. We found that enzyme activity was not reduced detectably in heterozygotes at any age, but was diminished greatly in homozygous mice compared with controls; thus, homozygous animals may suffer from a partial loss of function. The GarsC201R mutation described here is a contribution to our understanding of the mechanism by which mutations in tRNA synthetases, which are fundamentally important, ubiquitously expressed enzymes, cause axonopathy in specific sets of neurons.

Theoretical analyses of the Dendritic Cell Algorithm (DCA) have yielded several criticisms about its underlying structure and operation. As a result, several alterations and fixes have been suggested in the literature to correct for these findings. A contribution of this work is to investigate the effects of replacing the classification stage of the DCA (which is known to be flawed) with a traditional machine learning technique. This work goes on to question the merits of those unique properties of the DCA that are yet to be thoroughly analysed. If none of these properties can be found to have a benefit over traditional approaches, then \"fixing\" the DCA is arguably less efficient than simply creating a new algorithm. This work examines the dynamic filtering property of the DCA and questions the utility of this unique feature for the anomaly detection problem. It is found that this feature, while advantageous for noisy, time-ordered classification, is not as useful as a traditional static filter for processing a synthetic dataset. It is concluded that there are still unique features of the DCA left to investigate. Areas that may be of benefit to the Artificial Immune Systems community are suggested.

As one of the newest members in the field of artificial immune systems (AIS), the Dendritic Cell Algorithm (DCA) is based on behavioural models of natural dendritic cells (DCs). Unlike other AIS, the DCA does not rely on training data, instead domain or expert knowledge is required to predetermine the mapping between input signals from a particular instance to the three categories used by the DCA. This data preprocessing phase has received the criticism of having manually over-?tted the data to the algorithm, which is undesirable. Therefore, in this paper we have attempted to ascertain if it is possible to use principal component analysis (PCA) techniques to automatically categorise input data while still generating useful and accurate classication results. The integrated system is tested with a biometrics dataset for the stress recognition of automobile drivers. The experimental results have shown the application of PCA to the DCA for the purpose of automated data preprocessing is successful.

The dendritic cell algorithm is an immune-inspired technique for processing time-dependant data. Here we propose it as a possible solution for a robotic classification problem. The dendritic cell algorithm is implemented on a real robot and an investigation is performed into the effects of varying the migration threshold median for the cell population. The algorithm performs well on a classification task with very little tuning. Ways of extending the implementation to allow it to be used as a classifier within the field of robotic security are suggested.