A minimal CRISPR-Cas3 system for genome engineering, bioRxiv, 2019-12-03
AbstractCRISPR-Cas technologies have provided programmable gene editing tools that have revolutionized research. The leading CRISPR-Cas9 and Cas12a enzymes are ideal for programmed genetic manipulation, however, they are limited for genome-scale interventions. Here, we utilized a Cas3-based system featuring a processive nuclease, expressed endogenously or heterologously, for genome engineering purposes. Using an optimized and minimal CRISPR-Cas3 system (Type I-C) programmed with a single crRNA, large deletions ranging from 7 - 424 kb were generated in Pseudomonas aeruginosa with high efficiency and speed. By comparison, Cas9 yielded small deletions and point mutations. Cas3-generated deletion boundaries were variable in the absence of a homology-directed repair (HDR) template, and successfully and efficiently specified when present. The minimal Cas3 system is also portable; large deletions were induced with high efficiency in Pseudomonas syringae and Escherichia coli using an “all-in-one” vector. Notably, Cas3 generated bi-directional deletions originating from the programmed cut site, which was exploited to iteratively reduce a P. aeruginosa genome by 837 kb (13.5%) using 10 distinct crRNAs. We also demonstrate the utility of endogenous Cas3 systems (Type I-C and I-F) and develop an “anti-anti-CRISPR” strategy to circumvent endogenous CRISPR-Cas inhibitor proteins. CRISPR-Cas3 could facilitate rapid strain manipulation for synthetic biological and metabolic engineering purposes, genome minimization, and the analysis of large regions of unknown function.
biorxiv molecular-biology 0-100-users 2019Another look at microbe–metabolite interactions how scale invariant correlations can outperform a neural network, bioRxiv, 2019-12-02
AbstractMany scientists are now interested in studying the correlative relationships between microbes and metabolites. However, these kinds of analyses are complicated by the compositional (i.e., relative) nature of the data. Recently, Morton et al. proposed a neural network architecture called mmvec to predict metabolite abundances from microbe presence. They introduce this method as a scale invariant solution to the integration of multi-omics compositional data, and claim that “mmvec is the only method robust to scale deviations”. We do not doubt the utility of mmvec, but write in defense of simple linear statistics. In fact, when used correctly, correlation and proportionality can actually outperform the mmvec neural network.
biorxiv bioinformatics 0-100-users 2019Spontaneous generation of face recognition in untrained deep neural networks, bioRxiv, 2019-12-02
AbstractFace-selective neurons are observed in the primate visual pathway and are considered the basis of facial recognition in the brain. However, it is debated whether this neuronal selectivity can arise spontaneously, or requires training from visual experience. Here, we show that face-selective neurons arise spontaneously in random feedforward networks in the absence of learning. Using biologically inspired deep neural networks, we found that face-selective neurons arise under three different network conditions one trained using non-face natural images, one randomized after being trained, and one never trained. We confirmed that spontaneously emerged face-selective neurons show the biological view-point-invariant characteristics observed in monkeys. Such neurons suddenly vanished when feedforward weight variation declined to a certain level. Our results suggest that innate face-selectivity originates from statistical variation of the feedforward projections in hierarchical neural networks.
biorxiv neuroscience 0-100-users 2019The hematopoietic landscape at single-cell resolution reveals unexpected stem cell features in naked mole-rats, bioRxiv, 2019-12-02
SUMMARYNaked mole-rats are the longest-lived rodents endowed with resistance to cancer and age-related diseases, yet their stem cell characteristics remain enigmatic. We profiled the naked mole-rat hematopoietic system down to single-cell resolution, and identified several unique features likely contributing to longevity. In adult naked mole-rats red blood cells are formed in spleen and marrow, a neotenic feature beneficial for hypoxic environments and to prevent anemia. Platelet numbers are lower compared to short-lived mice, which may preclude age-related platelet increase and thrombosis. T cells mature in thymus and lymph nodes, providing a supply of T cells after age-related thymus involution. The pool of quiescent stem cells is higher than in mice, and HSCs overexpress an oxidative phosphorylation signature, revealing a new paradigm of stem cell metabolism to benefit longevity and oppose oncogenesis. Our work provides a platform to study immunology and stem cell biology in an animal model of healthy aging.HIGHLIGHTS<jatslist list-type=bullet><jatslist-item>Flow cytometry labelling panel to purify viable naked mole-rat HSPCs<jatslist-item><jatslist-item>The spleen as the major site of erythropoiesis in the naked mole-rat<jatslist-item><jatslist-item>Naked mole-rats show extrathymic T-cell development under homeostatic conditions<jatslist-item><jatslist-item>Naked mole-rat hematopoietic stem cells (HSCs) have high OXPHOS activity<jatslist-item>
biorxiv immunology 0-100-users 2019Frequent extrachromosomal oncogene amplification drives aggressive tumors, bioRxiv, 2019-11-29
Extrachromosomal DNA (ecDNA) amplification promotes high oncogene copy number, intratumoral genetic heterogeneity, and accelerated tumor evolution1–3, but its frequency and clinical impact are not well understood. Here we show, using computational analysis of whole-genome sequencing data from 1,979 cancer patients, that ecDNA amplification occurs in at least 26% of human cancers, of a wide variety of histological types, but not in whole blood or normal tissue. We demonstrate a highly significant enrichment for oncogenes on amplified ecDNA and that the most common recurrent oncogene amplifications arise on ecDNA. EcDNA amplifications resulted in higher levels of oncogene transcription compared to copy number matched linear DNA, coupled with enhanced chromatin accessibility. Patients whose tumors have ecDNA-based oncogene amplification showed increase of cell proliferation signature activity, greater likelihood of lymph node spread at initial diagnosis, and significantly shorter survival, even when controlled for tissue type, than do patients whose cancers are not driven by ecDNA-based oncogene amplification. The results presented here demonstrate that ecDNA-based oncogene amplification plays a central role in driving the poor outcome for patients with some of the most aggressive forms of cancers.
biorxiv cancer-biology 0-100-users 2019FMRP promotes RNA localization to neuronal projections through interactions between its RGG domain and G-quadruplex RNA sequences, bioRxiv, 2019-09-28
ABSTRACTThe sorting of RNA molecules to distinct subcellular locations facilitates the activity of spatially restricted processes through local protein synthesis. This process affects thousands of transcripts yet precisely how these RNAs are trafficked to their destinations remains generally unclear. Here we have analyzed subcellular transcriptomes of FMRP-null mouse neuronal cells to identify transcripts that depend on FMRP for efficient transport to neurites. We found that these FMRP RNA localization targets contain a large enrichment of G-quadruplex sequences, particularly in their 3′ UTRs, suggesting that FMRP recognizes these sequences to promote the localization of transcripts that contain them. Fractionation of neurons derived from human Fragile X Syndrome patients revealed a high degree of conservation in the identity of FMRP localization targets between human and mouse as well as an enrichment of G-quadruplex sequences in human FMRP RNA localization targets. Using high-throughput RNAprotein interaction assays and single-molecule RNA FISH, we identified the RGG domain of FMRP as important for both interaction with G-quadruplex RNA sequences and the neuronal transport of G-quadruplex-containing transcripts. Finally, we used ribosome footprinting to identify translational regulatory targets of FMRP. The translational regulatory targets were not enriched for G-quadruplex sequences and were largely distinct from the RNA localization targets of FMRP, indicating that the two functions can be biochemically separated and are mediated through different target recognition mechanisms. These results establish a molecular mechanism underlying FMRP-mediated neuronal RNA localization and provide a framework for the elucidation of similar mechanisms governed by other RNA-binding proteins.
biorxiv molecular-biology 0-100-users 2019