Aspergillus fumigatus is an opportunistic fungal pathogen that can cause life-threatening invasive lung infections in immunodeficient patients. The cellular and molecular processes of infection during onset, establishment, and progression of A. fumigatus infections are highly complex and depend on both fungal attributes and the immune status of the host. Therefore, preclinical animal models are of paramount importance to investigate and gain better insight into the infection process. Yet, despite their extensive use, commonly employed murine models of invasive pulmonary aspergillosis are not well understood due to analytical limitations. Here, we present quantitative light sheet fluorescence microscopy (LSFM) to describe fungal growth and the local immune response in whole lungs at cellular resolution within its anatomical context. We analyzed three very common murine models of pulmonary aspergillosis based on immunosuppression with corticosteroids, chemotherapy-induced leukopenia, or myeloablative irradiation. LSFM uncovered distinct architectures of fungal growth and degrees of tissue invasion in each model. Furthermore, LSFM revealed the spatial distribution, interaction, and activation of two key immune cell populations in antifungal defense: alveolar macrophages and polymorphonuclear neutrophils. Interestingly, the patterns of fungal growth correlated with the detected effects of the immunosuppressive regimens on the local immune cell populations. Moreover, LSFM demonstrates that the commonly used intranasal route of spore administration did not result in complete intra-alveolar deposition, as about 80% of fungal growth occurred outside the alveolar space. Hence, characterization by LSFM is more rigorous than by previously used methods employing murine models of invasive pulmonary aspergillosis and pinpoints their strengths and limitations. IMPORTANCE The use of animal models of infection is essential to advance our understanding of the complex host-pathogen interactions that take place during Aspergillus fumigatus lung infections. As in the case of humans, mice need to suffer an immune imbalance in order to become susceptible to invasive pulmonary aspergillosis (IPA), the most serious infection caused by A. fumigatus. There are several immunosuppressive regimens that are routinely used to investigate fungal growth and/or immune responses in murine models of invasive pulmonary aspergillosis. However, the precise consequences of the use of each immunosuppressive model for the local immune populations and for fungal growth are not completely understood. Here, to pin down the scenarios involving commonly used IPA models, we employed light sheet fluorescence microscopy (LSFM) to analyze whole lungs at cellular resolution. Our results will be valuable to optimize and refine animal models to maximize their use in future research.

Bacterial abortive-infection systems limit the spread of foreign invaders by shutting down or killing infected cells before the invaders can replicate1,2. Several RNA-targeting CRISPR–Cas systems (that is, types III and VI) cause abortive-infection phenotypes by activating indiscriminate nucleases3–5. However, a CRISPR-mediated abortive mechanism that leverages indiscriminate DNase activity of an RNA-guided single-effector nuclease has yet to be observed. Here we report that RNA targeting by the type V single-effector nuclease Cas12a2 drives abortive infection through non-specific cleavage of double-stranded DNA (dsDNA). After recognizing an RNA target with an activating protospacer-flanking sequence, Cas12a2 efficiently degrades single-stranded RNA (ssRNA), single-stranded DNA (ssDNA) and dsDNA. Within cells, the activation of Cas12a2 induces an SOS DNA-damage response and impairs growth, preventing the dissemination of the invader. Finally, we harnessed the collateral activity of Cas12a2 for direct RNA detection, demonstrating that Cas12a2 can be repurposed as an RNA-guided RNA-targeting tool. These findings expand the known defensive abilities of CRISPR–Cas systems and create additional opportunities for CRISPR technologies.

Bacterial genome editing commonly relies on chromosomal cleavage with Cas nucleases to counter-select against unedited cells. However, editing normally requires efficient recombination and high transformation efficiencies, which are unavailable in most strains. Here, we show that systematically attenuating DNA targeting activity enables RecA-mediated repair in different bacteria, allowing chromosomal cleavage to drive genome editing. Attenuation can be achieved by altering the format or expression strength of guide (g)RNAs; using nucleases with reduced cleavage activity; or engineering attenuated gRNAs (atgRNAs) with disruptive hairpins, perturbed nuclease-binding scaffolds, non-canonical PAMs, or guide mismatches. These modifications greatly increase cell counts and even improve the efficiency of different types of edits for Cas9 and Cas12a in Escherichia coli and Klebsiella oxytoca. We further apply atgRNAs to restore ampicillin sensitivity in Klebsiella pneumoniae, establishing a resistance marker for genetic studies. Attenuating DNA targeting thus offers a counterintuitive means to achieve CRISPR-driven editing across bacteria.

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SUMMARYImmune systems must recognize and clear foreign invaders without eliciting autoimmunity. CRISPR-Cas immune systems in prokaryotes manage this task by following two criteria: extensive guide:target complementarity and a defined target-flanking motif. Here we report an additional requirement for RNA-targeting CRISPR-Cas13 systems: expression of the target transcript exceeding a threshold. This finding is based on targeting endogenous non-essential transcripts, which rarely elicited dormancy through collateral RNA degradation. Instead, eliciting dormancy required over-expressing targeted transcripts above a threshold. A genome-wide screen confirmed target expression levels as the principal determinant of cytotoxic autoimmunity and revealed that the threshold shifts with the guide:target pair. This expression threshold ensured defense against a lytic bacteriophage yet allowed tolerance of a targeted beneficial gene expressed from an invading plasmid. These findings establish target expression levels as a third criterion for immune activation by RNA-targeting CRISPR-Cas systems, buffering against autoimmunity and distinguishing pathogenic and benign invaders.HIGHLIGHTSCas13-induced dormancy requires RNA target levels to exceed an expression thresholdThe expression threshold can prevent cytotoxic self-targeting for endogenous transcriptsThe threshold shifts depending on the CRISPR RNA guide:target pairThe threshold allows cells to distinguish pathogenic and benign infections

Bacterial genome editing commonly relies on chromosomal cleavage with Cas nucleases to counter-select against unedited cells. However, editing normally requires efficient recombination and high transformation efficiencies, which are unavailable in most strains. Here, we show that systematically attenuating DNA targeting activity enables RecA-mediated repair in different bacteria, allowing chromosomal cleavage to drive genome editing. Attenuation can be achieved by altering the format or expression strength of guide (g)RNAs; using nucleases with reduced cleavage activity; or engineering attenuated gRNAs (atgRNAs) with disruptive hairpins, perturbed nuclease scaffolds, non-canonical PAMs, or guide mismatches. These modifications greatly increase cell counts and even improve the efficiency of different types of edits for Cas9 and Cas12a in Escherichia coli and Klebsiella oxytoca. We further applied atgRNAs to restore ampicillin sensitivity in Klebsiella pneumoniae, establishing a new resistance marker for genetic studies. Attenuating DNA targeting thus offers a counterintuitive means to achieve CRISPR-driven editing across bacteria.

Bacterial abortive infection systems limit the spread of foreign invaders by shutting down or killing infected cells before the invaders can replicate. Several RNA-targeting CRISPR-Cas systems (e.g., types III and VI) cause Abi phenotypes by activating indiscriminate RNases. However, a CRISPR-mediated abortive mechanism that relies on indiscriminate DNase activity has yet to be observed. Here we report that RNA targeting by the type V Cas12a2 nuclease drives abortive infection through non-specific cleavage of double-stranded (ds)DNA. Upon recognition of an RNA target with an activating protospacer-flanking sequence, Cas12a2 efficiently degrades single–stranded (ss)RNA, ssDNA, and dsDNA. Within cells, the dsDNase activity induces an SOS response and impairs growth, stemming the infection. Finally, we harnessed the collateral activity of Cas12a2 for direct RNA detection, demonstrating that Cas12a2 can be repurposed as an RNA-guided, RNA-targeting tool. These findings expand the known defensive capabilities of CRISPR-Cas systems and create additional opportunities for CRISPR technologies.

Rudolf-Virchow-Zentrum für Experimentelle Biomedizin. 35 Universität Würzburg. 36 Josef-Schneider-Straße 2, Haus D15. D -97080 Würzburg 37 38 39 40 41 42 43 44 45 VISUAL ABSTRACT 46 Quantitative light sheet fluorescence microscopy to dissect local host-pathogen interactions 47 ABSTRACT 51Aspergillus fumigatus is an opportunistic fungal pathogen that can cause life-threatening 52 invasive lung infections in immunodeficient patients. The cellular and molecular processes of 53 infection during onset, establishment and progression are highly complex and depend on 54 both fungal attributes and the immune status of the host. Therefore, preclinical animal 55 models are paramount to investigate and gain better insight into the infection process. Yet, 56 despite their extensive use, commonly employed murine models of invasive pulmonary 57 aspergillosis are not well understood due to analytical limitations. Here we present 58 quantitative light sheet fluorescence microscopy (LSFM) to describe fungal growth and the 59 local immune response in whole lungs at cellular resolution within its anatomical context. We 60 analyzed three very common murine models of pulmonary aspergillosis based on 61 immunosuppression with corticosteroids, chemotherapy-induced leukopenia or 62 myeloablative irradiation. LSFM uncovered distinct architectures of fungal growth and 63 degrees of tissue invasion in each model. Furthermore, LSFM revealed the spatial 64 distribution, interaction and activation of two key immune cell populations in antifungal 65 defense: alveolar macrophages and polymorphonuclear neutrophils. Interestingly, the 66 patterns of fungal growth correlated with the detected effects of the immunosuppressive 67 regimens on the local immune cell populations. Moreover, LSFM demonstrates that the 68 commonly used intranasal route of spore administration did not result in the desired intra-69 alveolar deposition, as more than 60% of fungal growth occurred outside of the alveolar 70 space. Hence, LSFM allows for more rigorous characterization of murine models of invasive 71 pulmonary aspergillosis and pinpointing their strengths and limitations. 72 IMPORTANCE 73The use of animal models of infection is essential to advance our understanding of complex 74 host-pathogen interactions that take place during Aspergillus fumigatus lung infections. As in 75 the case of humans, mice need to be immunosuppressed to become susceptible to invasive 76 pulmonary aspergillosis, the most serious infection caused by A. fumigatus. There are 77 several immunosuppressive regimens that are routinely used to investigate fungal growth 78 and/or immune responses in murine models of invasive pulmonary aspergillosis (IPA). 79However, the precise consequences that each immunosuppressive model has on the local 80 immune populations and for fungal growth are not completely understood. Here we 81 employed light sheet fluorescence microscopy (LSFM) to analyze whole lungs at cellular 82 resolution, to pin down the scenario commonly used IPA models. Our results will be ...