Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes.

Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-AcrIIA15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13-AcrIIA15 as unique bifunctional inhibitors of SauCas9

GENE-43. TARGETING GABPb1L INHIBITS IN VIVO GROWTH OF TERT PROMOTER MUTANT GLIOBLASTOMA

Abstract Understanding cancer cell immortality in primary glioblastoma (GBM) is essential for the development of more informed treatments. Multiple cancer types, including >80% of GBMs, undergo immortalization by reactivating Telomerase Reverse Transcriptase (TERT) through acquired mutations in the TERT promoter. TERT, the catalytically active and rate-limiting subunit of telomerase, functions to maintain telomeres, which cap and protect the ends of chromosomes. Our past work has demonstrated that the transcription factor GABP - and specifically its tetramer-forming isoform GABPb1L - binds and activates the mutant TERT promoter. The generation of CRISPR-induced indels in GABPb1L results in a gradual loss of cell viability in TERT promoter mutant but not TERT promoter wild type tumor cells in vitro, but the extent to which GABPb1L function is compromised in this setting is unclear. Thus, the potential for use of GABPb1L as an effective therapeutic target for TERT promoter mutant GBM requires further investigation. Here, we use CRISPR-based strategies to demonstrate that full knockout of GABPb1L is rapidly lethal in TERT promoter mutant cells in vitro, in association with a decrease in both TERT mRNA and telomerase activity. Heterozygous deletion of GABPb1L in the context of TERT promoter mutations leads to slowed growth of orthotopic xenograft tumors in mice, and prolonged survival. Additionally, inducible RNAi-mediated inhibition of GABPb1L in growing tumors is also capable of decreasing tumor burden and increasing survival, further strongly suggesting that targeting GABPb1L in patient tumors could be a viable treatment strategy. Finally, reduced GABPb1L synergizes with temozolomide (TMZ) therapy such that TMZ treatment in the context of low GABPb1L and low TERT leads to a complete ablation of orthotopic GBM xenografts. These results highlight the potential to improve disease outcomes by targeting TERT through inhibition of GABPb1L, particularly in conjunction with TMZ treatment

GENE-43. TARGETING GABPb1L INHIBITS IN VIVO GROWTH OF TERT PROMOTER MUTANT GLIOBLASTOMA

Abstract Understanding cancer cell immortality in primary glioblastoma (GBM) is essential for the development of more informed treatments. Multiple cancer types, including >80% of GBMs, undergo immortalization by reactivating Telomerase Reverse Transcriptase (TERT) through acquired mutations in the TERT promoter. TERT, the catalytically active and rate-limiting subunit of telomerase, functions to maintain telomeres, which cap and protect the ends of chromosomes. Our past work has demonstrated that the transcription factor GABP - and specifically its tetramer-forming isoform GABPb1L - binds and activates the mutant TERT promoter. The generation of CRISPR-induced indels in GABPb1L results in a gradual loss of cell viability in TERT promoter mutant but not TERT promoter wild type tumor cells in vitro, but the extent to which GABPb1L function is compromised in this setting is unclear. Thus, the potential for use of GABPb1L as an effective therapeutic target for TERT promoter mutant GBM requires further investigation. Here, we use CRISPR-based strategies to demonstrate that full knockout of GABPb1L is rapidly lethal in TERT promoter mutant cells in vitro, in association with a decrease in both TERT mRNA and telomerase activity. Heterozygous deletion of GABPb1L in the context of TERT promoter mutations leads to slowed growth of orthotopic xenograft tumors in mice, and prolonged survival. Additionally, inducible RNAi-mediated inhibition of GABPb1L in growing tumors is also capable of decreasing tumor burden and increasing survival, further strongly suggesting that targeting GABPb1L in patient tumors could be a viable treatment strategy. Finally, reduced GABPb1L synergizes with temozolomide (TMZ) therapy such that TMZ treatment in the context of low GABPb1L and low TERT leads to a complete ablation of orthotopic GBM xenografts. These results highlight the potential to improve disease outcomes by targeting TERT through inhibition of GABPb1L, particularly in conjunction with TMZ treatment

A System-Level View on Out-of-Distribution Data in Robotics

When testing conditions differ from those represented in training data, so-called out-of-distribution (OOD) inputs can mar the reliability of black-box learned components in the modern robot autonomy stack. Therefore, coping with OOD data is an important challenge on the path towards trustworthy learning-enabled open-world autonomy. In this paper, we aim to demystify the topic of OOD data and its associated challenges in the context of data-driven robotic systems, drawing connections to emerging paradigms in the ML community that study the effect of OOD data on learned models in isolation. We argue that as roboticists, we should reason about the overall system-level competence of a robot as it performs tasks in OOD conditions. We highlight key research questions around this system-level view of OOD problems to guide future research toward safe and reliable learning-enabled autonomy

Characteristics of HIV-1 Discordant Couples Enrolled in a Trial of HSV-2 Suppression to Reduce HIV-1 Transmission: The Partners Study

Background: The Partners HSV-2/HIV-1 Transmission Study (Partners Study) is a phase III, placebo-controlled trial of daily acyclovir for genital herpes (HSV-2) suppression among HIV-1/HSV-2 co-infected persons to reduce HIV-1 transmission to their HIV-1 susceptible partners, which requires recruitment of HIV-1 serodiscordant heterosexual couples. We describe the baseline characteristics of this cohort. Methods: HIV-1 serodiscordant heterosexual couples, in which the HIV-1 infected partner was HSV-2 seropositive, had a CD4 count ≥250 cells/mcL and was not on antiretroviral therapy, were enrolled at 14 sites in East and Southern Africa. Demographic, behavioral, clinical and laboratory characteristics were assessed. Results: Of the 3408 HIV-1 serodiscordant couples enrolled, 67% of the HIV-1 infected partners were women. Couples had cohabitated for a median of 5 years (range 2–9) with 28% reporting unprotected sex in the month prior to enrollment. Among HIV-1 susceptible participants, 86% of women and 59% of men were HSV-2 seropositive. Other laboratory-diagnosed sexually transmitted infections were uncommon (500 relative to <350, respectively, p<0.001). Conclusions: The Partners Study successfully enrolled a cohort of 3408 heterosexual HIV-1 serodiscordant couples in Africa at high risk for HIV-1 transmission. Follow-up of this cohort will evaluate the efficacy of acyclovir for HSV-2 suppression in preventing HIV-1 transmission and provide insights into biological and behavioral factors determining heterosexual HIV-1 transmission. Trial Registration ClinicalTrials.gov NCT0019451

The JWST Early Release Science Program for Direct Observations of Exoplanetary Systems IV: NIRISS Aperture Masking Interferometry Performance and Lessons Learned

We present a performance analysis for the aperture masking interferometry (AMI) mode on board the James Webb Space Telescope Near Infrared Imager and Slitless Spectrograph (JWST/NIRISS). Thanks to self-calibrating observables, AMI accesses inner working angles down to and even within the classical diffraction limit. The scientific potential of this mode has recently been demonstrated by the Early Release Science (ERS) 1386 program with a deep search for close-in companions in the HIP 65426 exoplanetary system. As part of ERS 1386, we use the same dataset to explore the random, static, and calibration errors of NIRISS AMI observables. We compare the observed noise properties and achievable contrast to theoretical predictions. We explore possible sources of calibration errors, and show that differences in charge migration between the observations of HIP 65426 and point-spread function calibration stars can account for the achieved contrast curves. Lastly, we use self-calibration tests to demonstrate that with adequate calibration, NIRISS AMI can reach contrast levels of $\sim9-10$ mag. These tests lead us to observation planning recommendations and strongly motivate future studies aimed at producing sophisticated calibration strategies taking these systematic effects into account. This will unlock the unprecedented capabilities of JWST/NIRISS AMI, with sensitivity to significantly colder, lower mass exoplanets than ground-based setups at orbital separations inaccessible to JWST coronagraphy.Comment: 20 pages, 12 figures, submitted to AAS Journal

The \textit{JWST} Early Release Science Program for Direct Observations of Exoplanetary Systems III: Aperture Masking Interferometric Observations of the star HIP\,65426 at 3.8 μm\boldsymbol{3.8\,\rm{\mu m}}

We present aperture masking interferometry (AMI) observations of the star HIP 65426 at $3.8\,\rm{\mu m}$ as a part of the \textit{JWST} Direct Imaging Early Release Science (ERS) program obtained using the Near Infrared Imager and Slitless Spectrograph (NIRISS) instrument. This mode provides access to very small inner working angles (even separations slightly below the Michelson limit of ${}0.5\lambda/D$ for an interferometer), which are inaccessible with the classical inner working angles of the \textit{JWST} coronagraphs. When combined with \textit{JWST}'s unprecedented infrared sensitivity, this mode has the potential to probe a new portion of parameter space across a wide array of astronomical observations. Using this mode, we are able to achieve a contrast of $\Delta m_{F380M}{\sim }7.8$\,mag relative to the host star at a separation of {\sim}0.07\arcsec but detect no additional companions interior to the known companion HIP\,65426\,b. Our observations thus rule out companions more massive than 10{-}12\,\rm{M\textsubscript{Jup}} at separations ${\sim}10{-}20\,\rm{au}$ from HIP\,65426, a region out of reach of ground or space-based coronagraphic imaging. These observations confirm that the AMI mode on \textit{JWST} is sensitive to planetary mass companions orbiting at the water frost line, even for more distant stars at $\sim$100\,pc. This result will allow the planning and successful execution of future observations to probe the inner regions of nearby stellar systems, opening essentially unexplored parameter space.Comment: 15 pages, 9 figures, submitted to ApJ Letter