BackgroundNeurotropic virus-based tracers have been extensively applied in mapping and manipulation of neural circuits. However, their neurotropic and neurotoxic properties remain to be fully characterized.MethodsThrough neural circuit tracing, we systematically compared the neurotropism discrepancy among different multi-trans-synaptic and mono-synaptic retrograde viral tracers including pseudorabies virus (PRV), rabies virus (RV), and the newly engineered retro adeno-associated virus (rAAV2-retro) tracers. The (single-cell) RNA sequencing analysis was utilized for seeking possible attribution to neurotropism discrepancy and comparing cell toxicity caused by viral infection between glycoprotein-deleted RV (RV-∆G) and rAAV2-retro. Viral toxicity induced microglia activation and neuronal protein change were evaluated by immunohistochemistry.ResultsMulti-trans-synaptic retrograde viral tracers, PRV and RV, exhibit differential neurotropism when they were used for central neural circuit tracing from popliteal lymph nodes. Mono-synaptic retrograde tracers, including RV-∆G and rAAV2-retro, displayed discrepant neurotropic property, when they were applied to trace the inputs of lateral hypothalamic area and medial preoptic nucleus. rAAV2-retro demonstrated preference in cerebral cortex, whereas RV-∆G prefers to label basal ganglia and hypothalamus. Remarkably, we detected a distinct preference for specific cortical layer of rAAV2-retro in layer 5 and RV-∆G in layer 6 when they were injected into dorsal lateral geniculate nucleus to label corticothalamic neurons in primary visual cortex. Complementation of TVA receptor gene in RV-resistant neurons enabled EnvA-pseudotyped RV infection, supporting receptors attribution to viral neurotropism. Furthermore, both RV-∆G and rAAV2-retro exerted neurotoxic influence at the injection sites and retrogradely labeled sites, while the changes were more profound for RV-∆G infection. Finally, we demonstrated a proof-of-concept strategy for more comprehensive high-order circuit tracing of a specific target nucleus by combining rAAV2-retro, RV, and rAAV tracers.ConclusionsDifferent multi-trans-synaptic and mono-synaptic retrograde viral tracers exhibited discrepant neurotropism within certain brain regions, even cortical layer preference. More neurotoxicity was observed under RV-∆G infection as compared with rAAV2-retro. By combining rAAV2-retro, RV, and rAAV tracers, high-order circuit tracing can be achieved. Our findings provide important reference for appropriate application of viral tracers to delineate the landscape and dissect the function of neural network.Electronic supplementary materialThe online version of this article (10.1186/s13024-019-0308-6) contains supplementary material, which is available to authorized users.

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type III-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference (RNAi). This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single- and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.

Delineating the spatial multiomics landscape will pave the way to understanding the molecular basis of physiology and pathology. However, current spatial omics technology development is still in its infancy. Here, we developed a high-throughput multiomics in situ pairwise sequencing (MiP-Seq) strategy to efficiently decipher multiplexed DNAs, RNAs, proteins, and small biomolecules at subcellular resolution. We delineated dynamic spatial gene profiles in the hypothalamus using MiP-Seq. Moreover, MiP-Seq was unitized to detect tumor gene mutations and allele-specific expression of parental genes and to differentiate sites with and without the m6A RNA modification at specific sites. MiP-Seq was combined with in vivo Ca2+ imaging and Raman imaging to obtain a spatial multiomics atlas correlated to neuronal activity and cellular biochemical fingerprints. Importantly, we proposed a signal dilution strategy to resolve the crowded signals that challenge the applicability of in situ sequencing. Together, our method improves spatial multiomics and precision diagnostics and facilitates analyses of cell function in connection with gene profiles.

Tuberculosis caused by 15 Mycobacterium tuberculosis (M.tb) is the current leading infectious disease affecting more than 16 ten million people annually. To dissect the functional genomics and understand its virulence, 17 persistence, and antibiotics resistance, a powerful genome editing tool and high-throughput 18 screening methods are desperately wanted. Our study developed an efficient and a robust tool for 19 genome editing and RNA interference in M.tb using its endogenous CRISPR cas10 system. 20 Moreover, the system has been successfully applied for genome-wide CRISPR interference 21 screening. This tool could be employed to explore the functional genomics of M.tb and facilitate 22 the development of anti-M.tb drugs and vaccines. 23 Abstract: 24 Mycobacterium tuberculosis (M.tb) causes the current leading infectious disease. Examination of 25 the functional genomics of M.tb and development of drugs and vaccines are hampered by the 26 complicated and time-consuming genetic manipulation techniques for M.tb. Here, we 27 reprogrammed M.tb endogenous type III-A CRISPR-Cas10 system for simple and efficient gene 28 editing, RNA interference and screening via simple delivery of a plasmid harboring a 29 mini-CRISPR array, thereby avoiding the introduction of exogenous proteins and minimizing 30proteotoxicity. We demonstrated that M.tb genes were efficiently and specifically knocked-in/out 31 by this system, which was confirmed by whole-genome sequencing. This system was further 32 employed for single and simultaneous multiple-gene RNA interference. Moreover, we 33 successfully applied this system for genome-wide CRISPR interference screening to identify the 34 in-vitro and intracellular growth-regulating genes. This system can be extensively used to 35 explore the functional genomics of M.tb and facilitate the development of new 36 anti-Mycobacterial drugs and vaccines.37

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