Unsynchronized butyrophilin molecules dictate cancer cell evasion of Vγ9Vδ2 T-cell killing.

Vγ9Vδ2 T cells are specialized effector cells that have gained prominence as immunotherapy agents due to their ability to target and kill cells with altered pyrophosphate metabolites. In our effort to understand how cancer cells evade the cell-killing activity of Vγ9Vδ2 T cells, we performed a comprehensive genome-scale CRISPR screening of cancer cells. We found that four molecules belonging to the butyrophilin (BTN) family, specifically BTN2A1, BTN3A1, BTN3A2, and BTN3A3, are critically important and play unique, nonoverlapping roles in facilitating the destruction of cancer cells by primary Vγ9Vδ2 T cells. The coordinated function of these BTN molecules was driven by synchronized gene expression, which was regulated by IFN-γ signaling and the RFX complex. Additionally, an enzyme called QPCTL was shown to play a key role in modifying the N-terminal glutamine of these BTN proteins and was found to be a crucial factor in Vγ9Vδ2 T cell killing of cancer cells. Through our research, we offer a detailed overview of the functional genomic mechanisms that underlie how cancer cells escape Vγ9Vδ2 T cells. Moreover, our findings shed light on the importance of the harmonized expression and function of gene family members in modulating T-cell activity.

Strand-selective base editing of human mitochondrial DNA using mitoBEs.

A number of mitochondrial diseases in humans are caused by point mutations that could be corrected by base editors, but delivery of CRISPR guide RNAs into the mitochondria is difficult. In this study, we present mitochondrial DNA base editors (mitoBEs), which combine a transcription activator-like effector (TALE)-fused nickase and a deaminase for precise base editing in mitochondrial DNA. Combining mitochondria-localized, programmable TALE binding proteins with the nickase MutH or Nt.BspD6I(C) and either the single-stranded DNA-specific adenine deaminase TadA8e or the cytosine deaminase ABOBEC1 and UGI, we achieve A-to-G or C-to-T base editing with up to 77% efficiency and high specificity. We find that mitoBEs are DNA strand-selective mitochondrial base editors, with editing results more likely to be retained on the nonnicked DNA strand. Furthermore, we correct pathogenic mitochondrial DNA mutations in patient-derived cells by delivering mitoBEs encoded in circular RNAs. mitoBEs offer a precise, efficient DNA editing tool with broad applicability for therapy in mitochondrial genetic diseases.

CEA cell adhesion molecule 5 enriches functional human hematopoietic stem cells capable of long-term multi-lineage engraftment.

Hematopoietic stem cell (HSC) surface markers improve the understanding of cell identity and function. Here, we report that human HSCs can be distinguished by their expression of the CEA Cell Adhesion Molecule 5 (CEACAM5, CD66e), which serves as a marker and a regulator of HSC function. CD66e+ cells exhibited a 5.5-fold enrichment for functional long term HSCs compared to CD66e- cells. CD66e+CD34+CD90+CD45RA- cells displayed robust multi-lineage repopulation and serial reconstitution ability in immunodeficient mice compared to CD66e-CD34+CD90+CD45RA-cells. CD66e expression also identified almost all repopulating HSCs within the CD34+CD90+CD45RA- population. Together, these results indicated that CEACAM5 is a marker that enriches functional human hematopoietic stem cells capable of long-term multi-lineage engraftment.

Unbiased interrogation of functional lysine residues in human proteome.

CRISPR screens have empowered the high-throughput dissection of gene functions; however, more explicit genetic elements, such as codons of amino acids, require thorough interrogation. Here, we establish a CRISPR strategy for unbiasedly probing functional amino acid residues at the genome scale. By coupling adenine base editors and barcoded sgRNAs, we target 215,689 out of 611,267 (35%) lysine codons, involving 85% of the total protein-coding genes. We identify 1,572 lysine codons whose mutations perturb human cell fitness, with many of them implicated in cancer. These codons are then mirrored to gene knockout screen data to provide functional insights into the role of lysine residues in cellular fitness. Mining these data, we uncover a CUL3-centric regulatory network in which lysine residues of CUL3 CRL complex proteins control cell fitness by specifying protein-protein interactions. Our study offers a general strategy for interrogating genetic elements and provides functional insights into the human proteome.

Utilizing AAV-mediated LEAPER 2.0 for programmable RNA editing in non-human primates and nonsense mutation correction in humanized Hurler syndrome mice.

BACKGROUND: The endogenous adenosine deaminases acting on RNA (ADAR) have been harnessed to facilitate precise adenosine-to-inosine editing on RNAs. However, the practicability of this approach for therapeutic purposes is still ambiguous due to the variable expression of intrinsic ADAR across various tissues and species, as well as the absence of all-encompassing confirmation for delivery methods. RESULTS: In this study, we demonstrate that AAV-mediated delivery of circular ADAR-recruiting RNAs (arRNAs) achieves effective RNA editing in non-human primates at dosages suitable for therapy. Within a time frame of 4 to 13 weeks following infection, the editing efficiency in AAV-infected cells can reach approximately 80%, with no discernible toxicity, even at elevated dosages. In addition, when AAV-delivered circular arRNAs are systematically administered to a humanized mouse model of Hurler syndrome, it rectifies the premature stop codon precisely and restores the functionality of IDUA enzyme encoded by the Hurler causative gene in multiple organs. CONCLUSIONS: These discoveries considerably bolster the prospects of employing AAV-borne circular arRNAs for therapeutic applications and exploratory translational research.

DddA homolog search and engineering expand sequence compatibility of mitochondrial base editing.

Expanding mitochondrial base editing tools with broad sequence compatibility is of high need for both research and therapeutic purposes. In this study, we identify a DddA homolog from Simiaoa sunii (Ddd_Ss) which can efficiently deaminate cytosine in DC context in double-stranded DNA (dsDNA). We successfully develop Ddd_Ss-derived cytosine base editors (DdCBE_Ss) and introduce mutations at multiple mitochondrial DNA (mtDNA) loci including disease-associated mtDNA mutations in previously inaccessible GC context. Finally, by introducing a single amino acid substitution from Ddd_Ss, we successfully improve the activity and sequence compatibility of DdCBE derived from DddA of Burkholderia cenocepacia (DdCBE_Bc). Our study expands mtDNA editing tool boxes and provides resources for further screening and engineering dsDNA base editors for biological and therapeutic applications.

Human FcRn Is a Two-in-One Attachment-Uncoating Receptor for Echovirus 18.

Virus-receptor interactions determine viral host range and tissue tropism. CD55 and human neonatal Fc receptor (FcRn) were found to be the binding and uncoating receptors for some of the echovirus-related enterovirus species B serotypes in our previous study. Echovirus 18 (E18), as a member of enterovirus species B, is a significant causative agent of aseptic meningitis and viral encephalitis in children. However, it does not use CD55 as a critical host factor. We conducted CRISPR/Cas9 knockout screening to determine the receptors and entry mechanisms and identified FcRn working as a dual-function receptor for E18. Knockout of FCGRT and B2M, which encode the two subunits of FcRn, prevented infection by E18 and other echoviruses in the same physiological cluster. We then elucidated the underlying molecular mechanism of receptor recognition by E18 using cryogenic electron microscopy. The binding of the FCGRT subunit to the canyon region rotates the residues around the pocket, triggering the release of the pocket factor as observed for other enterovirus species B members. IMPORTANCE E18 is a member of enterovirus species B. As one of the most common enterovirus serotypes in nonpolio enterovirus detection, it easily infects children and causes various clinical symptoms. Aseptic meningitis and viral encephalitis are the most commonly reported syndromes associated with E18. No effective antiviral drugs or approved vaccines are available. Previous studies showed that CD55 and FcRn were the binding and uncoating receptors for some echoviruses. However, we found that CD55 is not the critical host factor for E18. Thus, we want to determine the receptors and elucidate the entry mechanism of E18. Our findings reveal that FcRn is a two-in-one attachment-uncoating receptor for E18.

Circular RNA vaccines against SARS-CoV-2 and emerging variants.

As the emerging variants of SARS-CoV-2 continue to drive the worldwide pandemic, there is a constant demand for vaccines that offer more effective and broad-spectrum protection. Here, we report a circular RNA (circRNA) vaccine that elicited potent neutralizing antibodies and T cell responses by expressing the trimeric RBD of the spike protein, providing robust protection against SARS-CoV-2 in both mice and rhesus macaques. Notably, the circRNA vaccine enabled higher and more durable antigen production than the 1mΨ-modified mRNA vaccine and elicited a higher proportion of neutralizing antibodies and distinct Th1-skewed immune responses. Importantly, we found that the circRNARBD-Omicron vaccine induced effective neutralizing antibodies against the Omicron but not the Delta variant. In contrast, the circRNARBD-Delta vaccine protected against both Delta and Omicron or functioned as a booster after two doses of either native- or Delta-specific vaccination, making it a favorable choice against the current variants of concern (VOCs) of SARS-CoV-2.

Regulatory elements can be essential for maintaining broad chromatin organization and cell viability.

Increasing evidence shows that promoters and enhancers could be related to 3D chromatin structure, thus affecting cellular functions. Except for their roles in forming canonical chromatin loops, promoters and enhancers have not been well studied regarding the maintenance of broad chromatin organization. Here, we focused on the active promoters/enhancers predicted to form many 3D contacts with other active promoters/enhancers (referred to as hotspots) and identified dozens of loci essential for cell growth and survival through CRISPR screening. We found that the deletion of an essential hotspot could lead to changes in broad chromatin organization and the expression of distal genes. We showed that the essentiality of hotspots does not result from their association with individual genes that are essential for cell viability but rather from their association with multiple dysregulated non-essential genes to synergistically impact cell fitness.

Gene editing and its applications in biomedicine.

The steady progress in genome editing, especially genome editing based on the use of clustered regularly interspaced short palindromic repeats (CRISPR) and programmable nucleases to make precise modifications to genetic material, has provided enormous opportunities to advance biomedical research and promote human health. The application of these technologies in basic biomedical research has yielded significant advances in identifying and studying key molecular targets relevant to human diseases and their treatment. The clinical translation of genome editing techniques offers unprecedented biomedical engineering capabilities in the diagnosis, prevention, and treatment of disease or disability. Here, we provide a general summary of emerging biomedical applications of genome editing, including open challenges. We also summarize the tools of genome editing and the insights derived from their applications, hoping to accelerate new discoveries and therapies in biomedicine.

Engineered circular ADAR-recruiting RNAs increase the efficiency and fidelity of RNA editing in vitro and in vivo.

Current methods for programmed RNA editing using endogenous ADAR enzymes and engineered ADAR-recruiting RNAs (arRNAs) suffer from low efficiency and bystander off-target editing. Here, we describe LEAPER 2.0, an updated version of LEAPER that uses covalently closed circular arRNAs, termed circ-arRNAs. We demonstrate on average ~3.1-fold higher editing efficiency than their linear counterparts when expressed in cells or delivered as in vitro-transcribed circular RNA oligonucleotides. To lower off-target editing we deleted pairings of uridines with off-target adenosines, which almost completely eliminated bystander off-target adenosine editing. Engineered circ-arRNAs enhanced the efficiency and fidelity of editing endogenous CTNNB1 and mutant TP53 transcripts in cell culture. Delivery of circ-arRNAs using adeno-associated virus in a mouse model of Hurler syndrome corrected the pathogenic point mutation and restored α-L-iduronidase catalytic activity, lowering glycosaminoglycan accumulation in the liver. LEAPER 2.0 provides a new design of arRNA that enables more precise, efficient RNA editing with broad applicability for therapy and basic research.

Genome-wide CRISPR activation screen identifies candidate receptors for SARS-CoV-2 entry.

The outbreak of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has created a global health crisis. SARS-CoV-2 infects varieties of tissues where the known receptor ACE2 is low or almost absent, suggesting the existence of alternative viral entry pathways. Here, we performed a genome-wide barcoded-CRISPRa screen to identify novel host factors that enable SARS-CoV-2 infection. Beyond known host proteins, i.e., ACE2, TMPRSS2, and NRP1, we identified multiple host components, among which LDLRAD3, TMEM30A, and CLEC4G were confirmed as functional receptors for SARS-CoV-2. All these membrane proteins bind directly to spike's N-terminal domain (NTD). Their essential and physiological roles have been confirmed in either neuron or liver cells. In particular, LDLRAD3 and CLEC4G mediate SARS-CoV-2 entry and infection in an ACE2-independent fashion. The identification of the novel receptors and entry mechanisms could advance our understanding of the multiorgan tropism of SARS-CoV-2, and may shed light on the development of COVID-19 countermeasures.

Low-density lipoprotein receptor-related protein 1 is a CROPs-associated receptor for Clostridioides infection toxin B.

As the leading cause of worldwide hospital-acquired infection, Clostridioides difficile (C. difficile) infection has caused heavy economic and hospitalized burden, while its pathogenesis is not fully understood. Toxin B (TcdB) is one of the major virulent factors of C. difficile. Recently, CSPG4 and FZD2 were reported to be the receptors that mediate TcdB cellular entry. However, genetic ablation of genes encoding these receptors failed to completely block TcdB entry, implicating the existence of alternative receptor(s) for this toxin. Here, by employing the CRISPR-Cas9 screen in CSPG4-deficient HeLa cells, we identified LDL receptor-related protein-1 (LRP1) as a novel receptor for TcdB. Knockout of LRP1 in both CSPG4-deficient HeLa cells and colonic epithelium Caco2 cells conferred cells with increased TcdB resistance, while LRP1 overexpression sensitized cells to TcdB at a low concentration. Co-immunoprecipitation assay showed that LRP1 interacts with full-length TcdB. Moreover, CROPs domain, which is dispensable for TcdB's interaction with CSPG4 and FZD2, is sufficient for binding to LRP1. As such, our study provided evidence for a novel mechanism of TcdB entry and suggested potential therapeutic targets for treating C. difficile infection.

Noncoding loci without epigenomic signals can be essential for maintaining global chromatin organization and cell viability.

Most noncoding regions of the human genome do not harbor any annotated element and are even not marked with any epigenomic or protein binding signal. However, an overlooked aspect of their possible role in stabilizing 3D chromatin organization has not been extensively studied. To illuminate their structural importance, we started with the noncoding regions forming many 3D contacts (referred to as hubs) and performed a CRISPR library screening to identify dozens of hubs essential for cell viability. Hi-C and single-cell transcriptomic analyses showed that their deletion could significantly alter chromatin organization and affect the expressions of distal genes. This study revealed the 3D structural importance of noncoding loci that are not associated with any functional element, providing a previously unknown mechanistic understanding of disease-associated genetic variations (GVs). Furthermore, our analyses also suggest a possible approach to develop therapeutics targeting disease-specific noncoding regions that are critical for disease cell survival.

Sensing of cytoplasmic chromatin by cGAS activates innate immune response in SARS-CoV-2 infection.

The global coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense RNA virus. How the host immune system senses and responds to SARS-CoV-2 infection remain largely unresolved. Here, we report that SARS-CoV-2 infection activates the innate immune response through the cytosolic DNA sensing cGAS-STING pathway. SARS-CoV-2 infection induces the cellular level of 2'3'-cGAMP associated with STING activation. cGAS recognizes chromatin DNA shuttled from the nucleus as a result of cell-to-cell fusion upon SARS-CoV-2 infection. We further demonstrate that the expression of spike protein from SARS-CoV-2 and ACE2 from host cells is sufficient to trigger cytoplasmic chromatin upon cell fusion. Furthermore, cytoplasmic chromatin-cGAS-STING pathway, but not MAVS-mediated viral RNA sensing pathway, contributes to interferon and pro-inflammatory gene expression upon cell fusion. Finally, we show that cGAS is required for host antiviral responses against SARS-CoV-2, and a STING-activating compound potently inhibits viral replication. Together, our study reported a previously unappreciated mechanism by which the host innate immune system responds to SARS-CoV-2 infection, mediated by cytoplasmic chromatin from the infected cells. Targeting the cytoplasmic chromatin-cGAS-STING pathway may offer novel therapeutic opportunities in treating COVID-19. In addition, these findings extend our knowledge in host defense against viral infection by showing that host cells' self-nucleic acids can be employed as a "danger signal" to alarm the immune system.

Proprotein convertase furina is required for heart development in zebrafish.

Cardiac looping and trabeculation are key processes during cardiac chamber maturation. However, the underlying mechanisms remain incompletely understood. Here, we report the isolation, cloning and characterization of the proprotein convertase furina from the cardiovascular mutant loft in zebrafish. loft is an ethylnitrosourea-induced mutant and has evident defects in the cardiac outflow tract, heart looping and trabeculation, the craniofacial region and pharyngeal arch arteries. Positional cloning revealed that furina mRNA was barely detectable in loft mutants, and loft failed to complement the TALEN-induced furina mutant pku338, confirming that furina is responsible for the loft mutant phenotypes. Mechanistic studies demonstrated that Notch reporter Tg(tp1:mCherry) signals were largely eliminated in mutant hearts, and overexpression of the Notch intracellular domain partially rescued the mutant phenotypes, probably due to the lack of Furina-mediated cleavage processing of Notch1b proteins, the only Notch receptor expressed in the heart. Together, our data suggest a potential post-translational modification of Notch1b proteins via the proprotein convertase Furina in the heart, and unveil the function of the Furina-Notch1b axis in cardiac looping and trabeculation in zebrafish, and possibly in other organisms.

Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs.

Canonical CRISPR-knockout (KO) screens rely on Cas9-induced DNA double-strand breaks (DSBs) to generate targeted gene KOs. These methodologies may yield distorted results because DSB-associated effects are often falsely assumed to be consequences of gene perturbation itself, especially when high copy-number sites are targeted. In the present study, we report a DSB-independent, genome-wide CRISPR screening method, termed iBARed cytosine base editing-mediated gene KO (BARBEKO). This method leverages CRISPR cytosine base editors for genome-scale KO screens by perturbing gene start codons or splice sites, or by introducing premature termination codons. Furthermore, it is integrated with iBAR, a strategy we devised for improving screening quality and efficiency. By constructing such a cell library through lentiviral infection at a high multiplicity of infection (up to 10), we achieved efficient and accurate screening results with substantially reduced starting cells. More importantly, in comparison with Cas9-mediated fitness screens, BARBEKO screens are no longer affected by DNA cleavage-induced cytotoxicity in HeLa-, K562- or DSB-sensitive retinal pigmented epithelial 1 cells. We anticipate that BARBEKO offers a valuable tool to complement the current CRISPR-KO screens in various settings.

TRIM26 is a critical host factor for HCV replication and contributes to host tropism.

Hepatitis C virus (HCV) remains a major human pathogen that requires better understanding of virus-host interactions. In this study, we performed a genome-wide CRISPR-Cas9 screening and identified TRIM26, an E3 ligase, as a critical HCV host factor. Deficiency of TRIM26 specifically impairs HCV genome replication. Mechanistic studies showed that TRIM26 interacts with HCV-encoded NS5B protein and mediates its K27-linked ubiquitination at residue K51, and thus promotes the NS5B-NS5A interaction. Moreover, mouse TRIM26 does not support HCV replication because of its unique six-amino acid insert that prevents its interaction with NS5B. Ectopic expression of human TRIM26 in a mouse hepatoma cell line that has been reconstituted with other essential HCV host factors promotes HCV infection. In conclusion, we identified TRIM26 as a host factor for HCV replication and a new determinant of host tropism. These results shed light on HCV-host interactions and may facilitate the development of an HCV animal model.

Multiscale Deficiency Integration by Na-Rich Engineering for High-Stability Li-Rich Layered Oxide Cathodes.

Lithium-rich manganese-based (LRM) layered oxides are considered as one of the most promising cathode materials for next-generation high-energy-density lithium-ion batteries (LIBs) because of their high specific capacity (>250 mAh g-1). However, they also go through severe capacity decay, serious voltage fading, and poor rate capability during cycling. Herein, a multiscale deficiency integration, including surface coating, subsurface defect construction, and bulk doping, is realized in a Li1.2Mn0.54Ni0.13Co0.13O2 cathode material by facile Na-rich engineering through a sol-gel method. This multiscale design can significantly improve the bulk and surface structural stability and diffusion rate of Li+ ions of electrode materials. Specifically, an outstanding specific capacity of 201 mAh g-1 is delivered at 1C of the designed cathode material after 400 cycles, relating to a large capacity retention of 89.0%. Meanwhile, the average voltage is retained up to 3.13 V with a large voltage retention of 89.6% and the energy density is maintained at 627.4 Wh kg-1. In situ X-ray diffraction (XRD), ex situ transmission electron microscopy (TEM) investigations, and density functional theory (DFT) calculations are conducted to explain the greatly enhanced electrochemical properties of a LRM cathode. We believe that this strategy would be a meaningful reference of LRM cathode materials for the research in the future.