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Type VI CRISPR enzymes cleave target RNAs and are widely used for gene regulation, RNA tracking, and diagnostics. However, a systematic understanding of their RNA binding specificity and cleavage activation is lacking. Here, we describeRNAchip-hybridizedassociation-mappingplatform (RNA-CHAMP), a massively parallel platform that repurposes next-generation DNA sequencing chips to measure the binding affinity for over 10,000 RNA targets containing structural perturbations, mismatches, insertions, and deletions relative to the CRISPR RNA (crRNA). Deep profiling of Cas13d, a compact and widely used RNA nuclease, reveals that it does not require a protospacer flanking sequence (PFS) but is exquisitely sensitive to secondary structure within the target RNA. Cas13d binding is strongly penalized by mismatches, insertions, and deletions in the distal crRNA-target RNA regions, while alterations in the proximal region inhibit nuclease activity without affecting binding. A biophysical model built from these data reveals that target recognition begins at the distal end of unstructured target RNAs and proceeds to the proximal end. Using this model, we designed a series of partially mismatched guide RNAs that modulate nuclease activity to detect single nucleotide polymorphisms (SNPs) in circulating SARS-CoV-2 variants. This work describes the key determinants of RNA targeting by a type VI CRISPR enzyme to improve CRISPR diagnostics andin vivoRNA editing. More broadly, RNA-CHAMP provides a quantitative platform for systematically measuring protein-RNA interactions.

DNA resection—the nucleolytic processing of broken DNA ends—is the first step of homologous recombination. Resection is catalyzed by the resectosome, a multi-enzyme complex that includes BLM helicase, DNA2 or EXO1 nucleases, and additional DNA-binding proteins. Although the molecular players have been known for over a decade, how the individual proteins work together to regulate DNA resection remain unknown. Using single-molecule imaging, we characterized the roles of MRN and TOP3A-RMI1/2 during long-range DNA resection. BLM partners with TOP3A-RMI1/2 to form the BTRR complex (or BLM dissolvasome). TOP3A-RMI1/2 aids BLM in initiating DNA unwinding, and along with MRN, stimulates DNA2-mediated resection. Furthermore, MRN promotes the association between BTRR and DNA, and synchronizes BLM and DNA2 translocation to prevent BLM from pausing during resection. Together, this work provides direct observation of how MRN and DNA2 harness the BTRR complex to resect DNA efficiently and how TOP3A-RMI1/2 regulates BLM’s helicase activity to promote efficient DNA repair.

DNA resection-the nucleolytic processing of broken DNA ends-is the first step of homologous recombination. Resection is catalyzed by the resectosome, a multi-enzyme complex that includes BLM helicase, DNA2 or EXO1 nucleases, and additional DNA-binding proteins. Although the molecular players have been known for over a decade, how the individual proteins work together to regulate DNA resection remains unknown. Using single-molecule imaging, we characterized the roles of the MRE11-RAD50-NBS1 complex (MRN) and Topoisomerase IIIa (TOP3A)-RMI1/2 during long-range DNA resection. BLM partners with TOP3A-RMI1/2 to form the BTRR complex (or BLM dissolvasome). We determined that TOP3A-RMI1/2 aids BLM in initiating DNA unwinding, and along with MRN, stimulates DNA2-mediated resection. Furthermore, we found that MRN promotes the association between BTRR and DNA, and synchronizes BLM and DNA2 translocation to prevent BLM from pausing during resection. Together, this work provides direct observation of how MRN and DNA2 harness the BTRR complex to resect DNA efficiently and how TOP3A-RMI1/2 regulates BLM's helicase activity to promote efficient DNA repair.

NanoCluster Beacons (NCBs) are multicolor silver nanocluster probes whose fluorescence can be activated or tuned by a proximal DNA strand called the activator. While a single-nucleotide difference in a pair of activators can lead to drastically different activation outcomes, termed the polar opposite twins (POTs), it is difficult to discover new POT-NCBs using the conventional low-throughput characterization approaches. Here we report a high-throughput selection method that takes advantage of repurposed next-generation-sequencing (NGS) chips to screen the activation fluorescence of ∼40,000 activator sequences. We find the nucleobases at positions 7-12 of the 18-nucleotide-long activator are critical to creating bright NCBs and positions 4-6 and 2-4 are hotspots to generate yellow and red POTs, respectively. Based on these findings, we propose a “zipper bag model” that explains how these hotspots lead to the creation of distinct silver cluster chromophores and contribute to the difference in chromophore chemical yields. Combining high-throughput screening with machine learning algorithms, we establish a pipeline to rationally design bright and multicolor NCBs.

The severe acute respiratory syndrome coronavirus 2 spike protein is a critical component of coronavirus disease 2019 vaccines and diagnostics and is also a therapeutic target. However, the spike protein is difficult to produce recombinantly because it is a large trimeric class I fusion membrane protein that is metastable and heavily glycosylated. We recently developed a prefusion-stabilized spike variant, termed HexaPro for six stabilizing proline substitutions, that can be expressed with a yield of >30 mg/L in ExpiCHO cells. This protocol describes an optimized workflow for expressing and biophysically characterizing rationally engineered spike proteins in Freestyle 293 and ExpiCHO cell lines. Although we focus on HexaPro, this protocol has been used to purify over a hundred different spike variants in our laboratories. We also provide guidance on expression quality control, long-term storage, and uses in enzyme-linked immunosorbent assays. The entire protocol, from transfection to biophysical characterization, can be completed in 7 d by researchers with basic tissue cell culture and protein purification expertise.

There is concern about second and subsequent waves of COVID-19 caused by the SARS-CoV-2 coronavirus occurring in communities globally that had an initial disease wave. Metropolitan Houston, TX, with a population of 7 million, is experiencing a massive second disease wave that began in late May 2020. To understand SARS-CoV-2 molecular population genomic architecture and evolution and the relationship between virus genotypes and patient features, we sequenced the genomes of 5,085 SARS-CoV-2 strains from these two waves. Our report provides the first molecular characterization of SARS-CoV-2 strains causing two distinct COVID-19 disease waves., We sequenced the genomes of 5,085 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains causing two coronavirus disease 2019 (COVID-19) disease waves in metropolitan Houston, TX, an ethnically diverse region with 7 million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston and from viruses recovered in an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently. Virtually all strains in the second wave have a Gly614 amino acid replacement in the spike protein, a polymorphism that has been linked to increased transmission and infectivity. Patients infected with the Gly614 variant strains had significantly higher virus loads in the nasopharynx on initial diagnosis. We found little evidence of a significant relationship between virus genotype and altered virulence, stressing the linkage between disease severity, underlying medical conditions, and host genetics. Some regions of the spike protein—the primary target of global vaccine efforts—are replete with amino acid replacements, perhaps indicating the action of selection. We exploited the genomic data to generate defined single amino acid replacements in the receptor binding domain of spike protein that, importantly, produced decreased recognition by the neutralizing monoclonal antibody CR3022. Our report represents the first analysis of the molecular architecture of SARS-CoV-2 in two infection waves in a major metropolitan region. The findings will help us to understand the origin, composition, and trajectory of future infection waves and the potential effect of the host immune response and therapeutic maneuvers on SARS-CoV-2 evolution.

Summary DNA repair deficiency leads to genome instability and hence human disease. Depletion of the RNA processing factor Y14/RBM8A in cultured cells or Rbm8a haplodeficiency in the developing mouse cortex results in the accumulation of DNA damage. Y14 depletion differentially affected the expression of DNA damage response (DDR) factors and induced R-loops, both of which threaten genomic stability. Immunoprecipitation coupled with mass spectrometry revealed DDR factors as potential Y14-interacting partners. Further results confirmed that Y14 interacts with Ku and several DDR factors in an ATM-dependent manner. Y14 co-fractionated with Ku in chromatin-enriched fractions and further accumulated on chromatin upon DNA damage. Y14 knockdown delayed recruitment of DDR factors to DNA damage sites and formation of γH2AX foci and also led to Ku retention on chromatin. Accordingly, Y14 depletion compromised the efficiency of DNA end joining. Therefore Y14 likely plays a direct role in DNA damage repair via its interaction with DDR factors., Graphical Abstract, Highlights • Y14 deficiency leads to DNA damage accumulation • Y14 depletion disturbs DNA damage response and induces R-loops • Y14 promotes Ku70/80 recruitment to DNA damage sites • Y14 participates in DNA damage repair, Biological Sciences; Molecular Biology; Cell Biology

Stabilizing the prefusion SARS-CoV-2 spike The development of therapeutic antibodies and vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is focused on the spike (S) protein that decorates the viral surface. A version of the spike ectodomain that includes two proline substitutions (S-2P) and stabilizes the prefusion conformation has been used to determine high-resolution structures. However, even S-2P is unstable and difficult to produce in mammalian cells. Hsieh et al. characterized many individual and combined structure-guided substitutions and identified a variant, named HexaPro, that retains the prefusion conformation but shows higher expression than S-2P and can also withstand heating and freezing. This version of the protein is likely to be useful in the development of vaccines and diagnostics. Science , this issue p. 1501

The SARS-CoV-2 spike protein is a critical component of vaccines and a target for neutralizing monoclonal antibodies (nAbs). Spike is also undergoing immunogenic selection with variants that increase infectivity and partially escape convalescent plasma. Here, we describe Spike Display, a high-throughput platform to rapidly characterize glycosylated spike ectodomains across multiple coronavirus-family proteins. We assayed ∼200 variant SARS-CoV-2 spikes for their expression, ACE2 binding, and recognition by 13 nAbs. An alanine scan of all five N-terminal domain (NTD) loops highlights a public epitope in the N1, N3, and N5 loops recognized by most NTD-binding nAbs. NTD mutations in variants of concern B.1.1.7 (alpha), B.1.351 (beta), B.1.1.28 (gamma), B.1.427/B.1.429 (epsilon), and B.1.617.2 (delta) impact spike expression and escape most NTD-targeting nAbs. Finally, B.1.351 and B.1.1.28 completely escape a potent ACE2 mimic. We anticipate that Spike Display will accelerate antigen design, deep scanning mutagenesis, and antibody epitope mapping for SARS-CoV-2 and other emerging viral threats., Graphical abstract, The SARS-CoV-2 spike protein is a critical target of the immune system. Javanmardi et al. report a mammalian cell display platform for rapidly screening the antigenicity and ACE2 affinity of spike variants. This platform, termed Spike Display, accelerates antigen design and antibody epitope mapping for viral glycoproteins.

Invented in 2010, NanoCluster Beacons (NCBs) (1) are an emerging class of turn-on probes that show unprecedented capabilities in single-nucleotide polymorphism (2) and DNA methylation (3) detection. As the activation colors of NCBs can be tuned by a near-by, guanine-rich activator strand, NCBs are versatile, multicolor probes suitable for multiplexed detection at low cost. Whereas a variety of NCB designs have been explored and reported, further diversification and optimization of NCBs require a full scan of the ligand composition space. However, the current methods rely on microarray and multi-well plate selection, which only screen tens to hundreds of activator sequences (4, 5). Here we take advantage of the next-generation-sequencing (NGS) platform for high-throughput, large-scale selection of activator strands. We first generated a ~104 activator sequence library on the Illumina MiSeq chip. Hybridizing this activator sequence library with a common nucleation sequence (which carried a nonfluorescent silver cluster) resulted in hundreds of MiSeq chip images with millions of bright spots (i.e. light-up polonies) of various intensities and colors. With a method termed Chip- Hybridized Associated Mapping Platform (CHAMP) (6), we were able to map these bright spots to the original DNA sequencing map, thus recovering the activator sequence behind each bright spot. After assigning an “activation score” to each “light-up polony”, we used a computational algorithm to select the best activator strands and validate these strands using the traditional in-solution preparation and fluorometer measurement method. By exploring a vast ligand composition space and observing the corresponding activation behaviors of silver clusters, we aim to elucidate the design rules of NCBs.

RBM4 modulates alternative exon selection of Numb and up-regulates proneural Mash1 gene expression, possibly via specific Numb isoforms. RBM4 overexpression promotes neuronal cell differentiation. Moreover, RBM4 is essential for neurite outgrowth in primary cortical neurons by modulating specific Numb isoform expression., RBM4 participates in cell differentiation by regulating tissue-specific alternative pre-mRNA splicing. RBM4 also has been implicated in neurogenesis in the mouse embryonic brain. Using mouse embryonal carcinoma P19 cells as a neural differentiation model, we observed a temporal correlation between RBM4 expression and a change in splicing isoforms of Numb, a cell-fate determination gene. Knockdown of RBM4 affected the inclusion/exclusion of exons 3 and 9 of Numb in P19 cells. RBM4-deficient embryonic mouse brain also exhibited aberrant splicing of Numb pre-mRNA. Using a splicing reporter minigene assay, we demonstrated that RBM4 promoted exon 3 inclusion and exon 9 exclusion. Moreover, we found that RBM4 depletion reduced the expression of the proneural gene Mash1, and such reduction was reversed by an RBM4-induced Numb isoform containing exon 3 but lacking exon 9. Accordingly, induction of ectopic RBM4 expression in neuronal progenitor cells increased Mash1 expression and promoted cell differentiation. Finally, we found that RBM4 was also essential for neurite outgrowth from cortical neurons in vitro. Neurite outgrowth defects of RBM4-depleted neurons were rescued by RBM4-induced exon 9–lacking Numb isoforms. Therefore our findings indicate that RBM4 modulates exon selection of Numb to generate isoforms that promote neuronal cell differentiation and neurite outgrowth.

Summary Genetic recombination in all kingdoms of life initiates when helicases and nucleases process (resect) the free DNA ends to expose single-stranded DNA (ssDNA) overhangs. Resection regulation in bacteria is programmed by a DNA sequence, but a general mechanism limiting resection in eukaryotes has remained elusive. Using single-molecule imaging of reconstituted human DNA repair factors, we identify phosphorylated RPA (pRPA) as a negative resection regulator. Bloom’s syndrome (BLM) helicase together with exonuclease 1 (EXO1) and DNA2 nucleases catalyze kilobase-length DNA resection on nucleosome-coated DNA. The resulting ssDNA is rapidly bound by RPA, which further stimulates DNA resection. RPA is phosphorylated during resection as part of the DNA damage response (DDR). Remarkably, pRPA inhibits DNA resection in cellular assays and in vitro via inhibition of BLM helicase. pRPA suppresses BLM initiation at DNA ends and promotes the intrinsic helicase strand-switching activity. These findings establish that pRPA provides a feedback loop between DNA resection and the DDR.