Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.

Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although most prokaryotic adaptive immune systems generally target DNA substrates, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase). How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear. Here we show that bacterial C2c2 possesses a unique RNase activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated single-stranded RNA degradation activity. These dual RNase functions are chemically and mechanistically different from each other and from the crRNA-processing behaviour of the evolutionarily unrelated CRISPR enzyme Cpf1 (ref. 11). The two RNase activities of C2c2 enable multiplexed processing and loading of guide RNAs that in turn allow sensitive detection of cellular transcripts.

Genomes in Focus: Development and Applications of CRISPR-Cas9 Imaging Technologies.

The discovery of the CRISPR-Cas9 endonuclease has enabled facile genome editing in living cells and organisms. Catalytically inactive Cas9 (dCas9) retains the ability to bind DNA in an RNA-guided fashion, and has additionally been explored as a tool for transcriptional modulation, epigenetic editing, and genome imaging. This Review highlights recent progress and challenges in the development of dCas9 for imaging genomic loci. The emergence and maturation of this technology offers the potential to answer mechanistic questions about chromosome dynamics and three-dimensional genome organization in vivo.

COVID-19 susceptibility and severity risks in a cross-sectional survey of over 500 000 US adults.

OBJECTIVES: The enormous toll of the COVID-19 pandemic has heightened the urgency of collecting and analysing population-scale datasets in real time to monitor and better understand the evolving pandemic. The objectives of this study were to examine the relationship of risk factors to COVID-19 susceptibility and severity and to develop risk models to accurately predict COVID-19 outcomes using rapidly obtained self-reported data. DESIGN: A cross-sectional study. SETTING: AncestryDNA customers in the USA who consented to research. PARTICIPANTS: The AncestryDNA COVID-19 Study collected self-reported survey data on symptoms, outcomes, risk factors and exposures for over 563 000 adult individuals in the USA in just under 4 months, including over 4700 COVID-19 cases as measured by a self-reported positive test. RESULTS: We replicated previously reported associations between several risk factors and COVID-19 susceptibility and severity outcomes, and additionally found that differences in known exposures accounted for many of the susceptibility associations. A notable exception was elevated susceptibility for men even after adjusting for known exposures and age (adjusted OR=1.36, 95% CI=1.19 to 1.55). We also demonstrated that self-reported data can be used to build accurate risk models to predict individualised COVID-19 susceptibility (area under the curve (AUC)=0.84) and severity outcomes including hospitalisation and critical illness (AUC=0.87 and 0.90, respectively). The risk models achieved robust discriminative performance across different age, sex and genetic ancestry groups within the study. CONCLUSIONS: The results highlight the value of self-reported epidemiological data to rapidly provide public health insights into the evolving COVID-19 pandemic.

Expanded COVID-19 phenotype definitions reveal distinct patterns of genetic association and protective effects.

Multiple COVID-19 genome-wide association studies (GWASs) have identified reproducible genetic associations indicating that there is a genetic component to susceptibility and severity risk. To complement these studies, we collected deep coronavirus disease 2019 (COVID-19) phenotype data from a survey of 736,723 AncestryDNA research participants. With these data, we defined eight phenotypes related to COVID-19 outcomes: four phenotypes that align with previously studied COVID-19 definitions and four 'expanded' phenotypes that focus on susceptibility given exposure, mild clinical manifestations and an aggregate score of symptom severity. We performed a replication analysis of 12 previously reported COVID-19 genetic associations with all eight phenotypes in a trans-ancestry meta-analysis of AncestryDNA research participants. In this analysis, we show distinct patterns of association at the 12 loci with the eight outcomes that we assessed. We also performed a genome-wide discovery analysis of all eight phenotypes, which did not yield new genome-wide significant loci but did suggest that three of the four 'expanded' COVID-19 phenotypes have enhanced power to capture protective genetic associations relative to the previously studied phenotypes. Thus, we conclude that continued large-scale ascertainment of deep COVID-19 phenotype data would likely represent a boon for COVID-19 therapeutic target identification.

Genome-wide analysis provides genetic evidence that ACE2 influences COVID-19 risk and yields risk scores associated with severe disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters human host cells via angiotensin-converting enzyme 2 (ACE2) and causes coronavirus disease 2019 (COVID-19). Here, through a genome-wide association study, we identify a variant (rs190509934, minor allele frequency 0.2-2%) that downregulates ACE2 expression by 37% (P = 2.7 × 10-8) and reduces the risk of SARS-CoV-2 infection by 40% (odds ratio = 0.60, P = 4.5 × 10-13), providing human genetic evidence that ACE2 expression levels influence COVID-19 risk. We also replicate the associations of six previously reported risk variants, of which four were further associated with worse outcomes in individuals infected with the virus (in/near LZTFL1, MHC, DPP9 and IFNAR2). Lastly, we show that common variants define a risk score that is strongly associated with severe disease among cases and modestly improves the prediction of disease severity relative to demographic and clinical factors alone.

Mechanistic study of the stereoselective polymerization of D,L-lactide using indium(III) halides.

We report the results of a comprehensive investigation of the recently discovered stereoselective and controlled polymerization of racemic lactide (D,L-LA) using an initiator prepared in situ from indium(III) chloride (InCl(3)), benzyl alcohol (BnOH), and triethylamine (NEt(3)). Linear relationships between number-average molecular weight (M(n)) and both monomer to alcohol concentration ratio and monomer conversion are consistent with a well-controlled polymerization. Studies on polymerization kinetics show the process to be first-order in [InCl(3)](0) and zero-order in both [BnOH](0) and [NEt(3)](0). The rate of D,L-LA conversion is also dependent on the indium(III) halide (i.e., t(1/2)(InCl(3)) approximately = 43 min versus t(1/2)(InBr(3)) approximately = 7.5 h, 21 degrees C, CD(2)Cl(2), [D,L-LA](0)/[BnOH](0) approximately = 100, [D,L-LA](0) = 0.84 M, [InX(3)](0)/[BnOH](0) = 1) and lactide stereoisomer (i.e., k(obs)(D,L-LA) approximately = k(obs)(meso-LA) > k(obs)(L-LA)). A model system that polymerizes D,L-LA with the same high degree of stereoselectivity was developed using 3-diethylamino-1-propanol (deapH) in lieu of BnOH and NEt(3). The product of the reaction of deapH with InCl(3) was identified as [InCl(3)(deapH)(H(2)O)](2) by elemental analysis, X-ray crystallography, and NMR and FTIR spectroscopies. An anhydrous version of the complex was also isolated when care was taken to avoid adventitious water, and was shown by pulsed gradient spin-echo (PGSE) NMR experiments to adopt a dinuclear structure in CD(2)Cl(2) solution under conditions identical to those used in its stereoselective polymerization of D,L-LA. The combined data suggest that the initiating species for the InCl(3)/BnOH/NEt(3) system is similar to [InCl(3)(deapH)(H(2)O)](2) and of the type [InCl((3-n))(OBn)(n)](m). With this information we propose a mechanism that rationalizes the observed stereocontrol in D,L-LA polymerizations. Finally, in an exploration of the scope of the InCl(3)/BnOH/NEt(3) system, we found this system to be effective for the polymerization of other cyclic esters, including epsilon-caprolactone and several substituted derivatives.

Dynamics of CRISPR-Cas9 genome interrogation in living cells.

The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.