Programmable double-strand DNA breaks (DSBs) can be harnessed for precision genome editing through manipulation of the homology-directed repair (HDR) pathway. However, end-joining repair pathways ...often outcompete HDR and introduce insertions and deletions of bases (indels) at the DSB site, decreasing precision outcomes. It has been shown that indel sequences for a given DSB site are reproducible and can even be predicted. Here, we report a general strategy (the "double tap" method) to improve HDR-mediated precision genome editing efficiencies that takes advantage of the reproducible nature of indel sequences. The method simply involves the use of multiple gRNAs: a primary gRNA that targets the wild-type genomic sequence, and one or more secondary gRNAs that target the most common indel sequence(s), which in effect provides a "second chance" at HDR-mediated editing. This proof-of-principle study presents the double tap method as a simple yet effective option for enhancing precision editing in mammalian cells.
A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families that are known to be produced by the microbiome have a marked effect ...on the balance between health and disease
. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units
), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches
to provide an assessment of the effects of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free and specific-pathogen-free mice. We found that the microbiota affects the chemistry of all organs. This included the amino acid conjugations of host bile acids that were used to produce phenylalanocholic acid, tyrosocholic acid and leucocholic acid, which have not previously been characterized despite extensive research on bile-acid chemistry
. These bile-acid conjugates were also found in humans, and were enriched in patients with inflammatory bowel disease or cystic fibrosis. These compounds agonized the farnesoid X receptor in vitro, and mice gavaged with the compounds showed reduced expression of bile-acid synthesis genes in vivo. Further studies are required to confirm whether these compounds have a physiological role in the host, and whether they contribute to gut diseases that are associated with microbiome dysbiosis.
Homing CRISPR gene drives could aid in curbing the spread of vector-borne diseases and controlling crop pest and invasive species populations due to an inheritance rate that surpasses Mendelian laws. ...However, this technology suffers from resistance alleles formed when the drive-induced DNA break is repaired by error-prone pathways, which creates mutations that disrupt the gRNA recognition sequence and prevent further gene-drive propagation. Here, we attempt to counteract this by encoding additional gRNAs that target the most commonly generated resistance alleles into the gene drive, allowing a second opportunity at gene-drive conversion. Our presented "double-tap" strategy improved drive efficiency by recycling resistance alleles. The double-tap drive also efficiently spreads in caged populations, outperforming the control drive. Overall, this double-tap strategy can be readily implemented in any CRISPR-based gene drive to improve performance, and similar approaches could benefit other systems suffering from low HDR frequencies, such as mammalian cells or mouse germline transformations.
Re-operation for positive resection margins following breast-conserving surgery occurs frequently (average = 20-25%), is cost-inefficient, and leads to physical and psychological morbidity. Current ...margin assessment techniques are slow and labour intensive. Rapid evaporative ionisation mass spectrometry (REIMS) rapidly identifies dissected tissues by determination of tissue structural lipid profiles through on-line chemical analysis of electrosurgical aerosol toward real-time margin assessment.
Electrosurgical aerosol produced from ex-vivo and in-vivo breast samples was aspirated into a mass spectrometer (MS) using a monopolar hand-piece. Tissue identification results obtained by multivariate statistical analysis of MS data were validated by histopathology. Ex-vivo classification models were constructed from a mass spectral database of normal and tumour breast samples. Univariate and tandem MS analysis of significant peaks was conducted to identify biochemical differences between normal and cancerous tissues. An ex-vivo classification model was used in combination with bespoke recognition software, as an intelligent knife (iKnife), to predict the diagnosis for an ex-vivo validation set. Intraoperative REIMS data were acquired during breast surgery and time-synchronized to operative videos.
A classification model using histologically validated spectral data acquired from 932 sampling points in normal tissue and 226 in tumour tissue provided 93.4% sensitivity and 94.9% specificity. Tandem MS identified 63 phospholipids and 6 triglyceride species responsible for 24 spectral differences between tissue types. iKnife recognition accuracy with 260 newly acquired fresh and frozen breast tissue specimens (normal n = 161, tumour n = 99) provided sensitivity of 90.9% and specificity of 98.8%. The ex-vivo and intra-operative method produced visually comparable high intensity spectra. iKnife interpretation of intra-operative electrosurgical vapours, including data acquisition and analysis was possible within a mean of 1.80 seconds (SD ±0.40).
The REIMS method has been optimised for real-time iKnife analysis of heterogeneous breast tissues based on subtle changes in lipid metabolism, and the results suggest spectral analysis is both accurate and rapid. Proof-of-concept data demonstrate the iKnife method is capable of online intraoperative data collection and analysis. Further validation studies are required to determine the accuracy of intra-operative REIMS for oncological margin assessment.
Clearance of surgical margins in cervical cancer prevents the need for adjuvant chemoradiation and allows fertility preservation. In this study, we determined the capacity of the rapid evaporative ...ionization mass spectrometry (REIMS), also known as intelligent knife (iKnife), to discriminate between healthy, preinvasive, and invasive cervical tissue. Cervical tissue samples were collected from women with healthy, human papilloma virus (HPV) ± cervical intraepithelial neoplasia (CIN), or cervical cancer. A handheld diathermy device generated surgical aerosol, which was transferred into a mass spectrometer for subsequent chemical analysis. Combination of principal component and linear discriminant analysis and least absolute shrinkage and selection operator was employed to study the spectral differences between groups. Significance of discriminatory m/z features was tested using univariate statistics and tandem MS performed to elucidate the structure of the significant peaks allowing separation of the two classes. We analyzed 87 samples (normal = 16, HPV ± CIN = 50, cancer = 21 patients). The iKnife discriminated with 100% accuracy normal (100%) vs. HPV ± CIN (100%) vs. cancer (100%) when compared to histology as the gold standard. When comparing normal vs. cancer samples, the accuracy was 100% with a sensitivity of 100% (95% CI 83.9 to 100) and specificity 100% (79.4 to 100). Univariate analysis revealed significant MS peaks in the cancer-to-normal separation belonging to various classes of complex lipids. The iKnife discriminates healthy from premalignant and invasive cervical lesions with high accuracy and can improve oncological outcomes and fertility preservation of women treated surgically for cervical cancer. Larger in vivo research cohorts are required to validate these findings.
Volatile aldehydes are enriched in esophageal adenocarcinoma (EAC) patients' breath and could improve early diagnosis, however the mechanisms of their production are unknown. Here, we show that weak ...aldehyde detoxification characterizes EAC, which is sufficient to cause endogenous aldehyde accumulation in vitro. Two aldehyde groups are significantly enriched in EAC biopsies and adjacent tissue: (i) short-chain alkanals, and (ii) medium-chain alkanals, including decanal. The short-chain alkanals form DNA-adducts, which demonstrates genotoxicity and confirms inadequate detoxification. Metformin, a putative aldehyde scavenger, reduces this toxicity. Tissue and breath concentrations of the medium-chain alkanal decanal are correlated, and increased decanal is linked to reduced ALDH3A2 expression, TP53 deletion, and adverse clinical features. Thus, we present a model for increased exhaled aldehydes based on endogenous accumulation from reduced detoxification, which also causes therapeutically actionable genotoxicity. These results support EAC early diagnosis trials using exhaled aldehyde analysis.
The accurate and timely identification of the causative organism of infection is important in ensuring the optimum treatment regimen is prescribed for a patient. Rapid evaporative ionisation mass ...spectrometry (REIMS), using electrical diathermy for the thermal disruption of a sample, has been shown to provide fast and accurate identification of microorganisms directly from culture. However, this method requires contact to be made between the REIMS probe and microbial biomass; resulting in the necessity to clean or replace the probes between analyses. Here, optimisation and utilisation of ambient laser desorption ionisation (ALDI) for improved speciation accuracy and analytical throughput is shown. Optimisation was completed on 15 isolates of Escherichia coli, showing 5 W in pulsatile mode produced the highest signal-to-noise ratio. These parameters were used in the analysis of 150 clinical isolates from ten microbial species, resulting in a speciation accuracy of 99.4% - higher than all previously reported REIMS modalities. Comparison of spectral data showed high levels of similarity between previously published electrical diathermy REIMS data. ALDI does not require contact to be made with the sample during analysis, meaning analytical throughput can be substantially improved, and further, increases the range of sample types which can be analysed in potential direct-from-sample pathogen detection.
Rapid evaporative ionization mass spectrometry (REIMS) was used for the rapid mass spectrometric profiling of cancer cell lines. Spectral reproducibility was assessed for three different cell lines, ...and the extent of interclass differences and intraclass variance was found to allow the identification of these cell lines based on the REIMS data. Subsequently, the NCI60 cell line panel was subjected to REIMS analysis, and the resulting data set was investigated for its distinction of individual cell lines and different tissue types of origin. Information content of REIMS spectral profiles of cell lines were found to be similar to those obtained from mammalian tissues although pronounced differences in relative lipid intensity were observed. Ultimately, REIMS was shown to detect changes in lipid content of cell lines due to mycoplasma infection. The data show that REIMS is an attractive means to study cell lines involving minimal sample preparation and analysis times in the range of seconds.
Delays in the diagnosis and treatment of endometrial cancer negatively impact patient survival. The aim of this study was to establish whether rapid evaporative ionisation mass spectrometry using the ...iKnife can accurately distinguish between normal and malignant endometrial biopsy tissue samples in real time, enabling point-of-care (POC) diagnoses.
Pipelle biopsy samples were obtained from consecutive women needing biopsies for clinical reasons. A Waters G2-XS Xevo Q-Tof mass spectrometer was used in conjunction with a modified handheld diathermy (collectively called the 'iKnife'). Each tissue sample was processed with diathermy, and the resultant surgical aerosol containing ionic lipid species was then analysed, producing spectra. Principal component analyses and linear discriminant analyses were performed to determine variance in spectral signatures. Leave-one-patient-out cross-validation was used to test the diagnostic accuracy.
One hundred and fifty patients provided Pipelle biopsy samples (85 normal, 59 malignant, 4 hyperplasia and 2 insufficient), yielding 453 spectra. The iKnife differentiated between normal and malignant endometrial tissues on the basis of differential phospholipid spectra. Cross-validation revealed a diagnostic accuracy of 89% with sensitivity, specificity, positive predictive value and negative predictive value of 85%, 93%, 94% and 85%, respectively.
This study is the first to use the iKnife to identify cancer in endometrial Pipelle biopsy samples. These results are highly encouraging and suggest that the iKnife could be used in the clinic to provide a POC diagnosis.
In this study, we integrate rapid evaporative ionization mass spectrometry (REIMS) with the Harmonic scalpel, an advanced laparoscopic surgical instrument that utilizes ultrasound energy to dissect ...and coagulate tissues. It provides unparalleled manipulation capability to surgeons and has superseded traditional electrosurgical tools particularly in abdominal surgery, but is yet to be validated with REIMS. The REIMS platform coupled with the Harmonic device was shown to produce tissue-specific lipid profiles through the analysis of porcine tissues in both negative and positive ionization modes. Comparison with other methods of electrosurgical dissection, such as monopolar electrosurgery and CO2 laser, showed spectral differences in the profile dependent on the energy device used. The Harmonic device demonstrated major spectral differences in the phospholipid region of m/z 600–1000 compared with the monopolar electrosurgical and CO2 laser-generated spectra. Within the Harmonic REIMS spectra, high intensities of diglycerides and triglycerides were observed. In contrast, monopolar electrosurgical and laser spectra demonstrated high abundances of glycerophospholipids. The Harmonic scalpel was able to differentiate between the liver, muscle, colon, and small intestine, demonstrating 100% diagnostic accuracy. The validation of the Harmonic device–mass spectrometry combination will allow the platform to be used safely and robustly for real-time in vivo surgical tissue identification in a variety of clinical applications.
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