The increasing use of CRISPR–Cas9 in medicine, agriculture, and synthetic biology has accelerated the drive to discover new CRISPR–Cas inhibitors as potential mechanisms of control for gene editing applications. Many anti-CRISPRs have been found that inhibit the CRISPR–Cas adaptive immune system. However, comparing all currently known anti-CRISPRs does not reveal a shared set of properties for facile bioinformatic identification of new anti-CRISPR families. Here, we describe AcRanker, a machine learning based method to aid direct identification of new potential anti-CRISPRs using only protein sequence information. Using a training set of known anti-CRISPRs, we built a model based on XGBoost ranking. We then applied AcRanker to predict candidate anti-CRISPRs from predicted prophage regions within self-targeting bacterial genomes and discovered two previously unknown anti-CRISPRs: AcrllA20 (ML1) and AcrIIA21 (ML8). We show that AcrIIA20 strongly inhibits Streptococcus iniae Cas9 (SinCas9) and weakly inhibits Streptococcus pyogenes Cas9 (SpyCas9). We also show that AcrIIA21 inhibits SpyCas9, Streptococcus aureus Cas9 (SauCas9) and SinCas9 with low potency. The addition of AcRanker to the anti-CRISPR discovery toolkit allows researchers to directly rank potential anti-CRISPR candidate genes for increased speed in testing and validation of new anti-CRISPRs. A web server implementation for AcRanker is available online at http://acranker.pythonanywhere.com/.

Highlights d Cryo-EM reveals the molecular features of the mammalian SRP targeting complexes d The fold of the eukaryotic SRP54 M domain and signal sequence binding is revealed d SRP$SR complex reveals eukaryotic-specific SRP contacts at the distal site d Regulation of the SRP$SR GTPase occurs through conserved protein and RNA contacts

CD8 T cells are necessary for the elimination of intracellular pathogens, but during chronic viral infections, CD8 T cells become exhausted and unable to control the persistent infection. Programmed cell death-1 (PD-1) blockade therapies have been shown to improve CD8 T cell responses during chronic viral infections. These therapies have been licensed to treat cancers in humans, but they have not yet been licensed to treat chronic viral infections because limited benefit is seen in pre-clinical animal models of chronic infection. In the present study, we investigated whether TLR4 triggering could improve PD-1 therapy during a chronic viral infection. Using the model of chronic lymphocytic choriomeningitis virus (LCMV) infection in mice, we show that TLR4 triggering with sublethal doses of lipopolysaccharide (LPS) followed by PD-1 blockade results in superior improvement in circulating virus-specific CD8 T cell responses, relative to PD-1 blockade alone. Moreover, we show that the synergy between LPS and PD-1 blockade is dependent on B7 costimulation and mediated by a dendritic cell (DC) intrinsic mechanism. Systemic LPS administration may have safety concerns, motivating us to devise a safer regimen. We show that ex vivo activation of DCs with LPS, followed by adoptive DC transfer, results in a similar potentiation of PD-1 therapy without inducing wasting disease. In summary, our data demonstrate a previously unidentified role for LPS/TLR4 signaling in modulating the host response to PD-1 therapy. These findings may be important for developing novel checkpoint therapies against chronic viral infection.

ABSTRACTThe increasing use of CRISPR-Cas9 in medicine, agriculture and synthetic biology has accelerated the drive to discover new CRISPR-Cas inhibitors as potential mechanisms of control for gene editing applications. Many such anti-CRISPRs have been found in mobile genetic elements that disable the CRISPR-Cas adaptive immune system. However, comparing all currently known anti-CRISPRs does not reveal a shared set of properties that can be used for facile bioinformatic identification of new anti-CRISPR families. Here, we describe AcRanker, a machine learning based method for identifying new potential anti-CRISPRs directly from proteomes using protein sequence information only. Using a training set of known anti-CRISPRs, we built a model based on XGBoost ranking and extensively benchmarked it through non-redundant cross-validation and external validation. We then applied AcRanker to predict candidate anti-CRISPRs from self-targeting bacterial genomes and discovered two previously unknown anti-CRISPRs: AcrllA16 (ML1) and AcrIIA17 (ML8). We show that AcrIIA16 strongly inhibits Streptococcus iniae Cas9 (SinCas9) and weakly inhibits Streptococcus pyogenes Cas9 (SpyCas9). We also show that AcrIIA17 inhibits both SpyCas9 and SauCas9 with low potency. The addition of AcRanker to the anti-CRISPR discovery toolkit allows researchers to directly rank potential anti-CRISPR candidate genes for increased speed in testing and validation of new anti-CRISPRs. A web server implementation for AcRanker is available online at http://acranker.pythonanywhere.com/.

CD8 T cells are critical for controlling intracellular pathogens, but during chronic viral infection, these cells become exhausted and unable to control virus. Programmed cell death-1 (PD-1) blockade therapy partially improves exhausted CD8 T cells during chronic viral infections. However, its limited efficacy in models of chronic infection has precluded its licensure to treat chronic infection. Interestingly, prior studies show that certain microbiota can improve PD-1 therapy, but the specific microbial components that mediate this positive effect remain unknown. In this study, we interrogated whether microbial LPS could enhance PD-1 therapy during chronic viral infection. Using the model of chronic lymphocytic choriomeningitis virus (LCMV) infection in mice, we show that sublethal doses of LPS followed by PD-1 therapy results in synergistic improvement of virus-specific CD8 T cells in blood and tissues (~10-fold greater compared to PD-1 therapy alone). We show that the improvement of PD-1 therapy by LPS is dependent on reinforced B7/CD28 costimulation and is mediated by a dendritic cell intrinsic mechanism. Although LPS has safety concerns, prior studies show that low LPS doses (≤4 ng/kg) are well-tolerated in humans, demonstrating that there is a threshold of natural resistance to this microbial product. Altogether, our data suggest an unexpected role for LPS/TLR4 signaling in modulating response to PD-1 therapy. Our findings may be important for improving immune checkpoint therapy against chronic infections and for understanding how microbial products modulate responses to therapy.

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