Biology subjects

Tian, B.

Publications and source records attributed to Tian, B..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

The m6A reader IGF2BP3 promotes gastric cancer progression via increasing the expression of FBXO32 protein

N6-methyladenosine (m6A) represents the most prevalent chemical modification on eukaryotic mRNA, with an accumulating body of literature indicating its pivotal significance in the pathogenesis of human cancers. Nevertheless, the precise molecular interplay between the m6A reader protein IGF2BP3 and gastric cancer remains to be thoroughly delineated. Our study uncovered that the expression of IGF2BP3 in gastric cancer tissues is markedly elevated in comparison to adjacent normal tissues, and this upregulation is tightly correlated with the incidence of lymph node metastasis, more advanced TNM stages, and deeper invasion depth of tumor in patients. In vitro experiments demonstrated that IGF2BP3 potentiates the proliferative, migratory, and invasive capacities of gastric cancer cells, while concurrently inhibiting apoptosis and augmenting the intracellular levels of aerobic glycolysis. In vivo experiments revealed that IGF2BP3 contributes to the growth of gastric cancer. Mechanistically, IGF2BP3 can increase the expression of FBXO32 protein by recognizing and binding to the m6A binding site on FBXO32 mRNA and further activate the downstream cGMP-PKG signaling pathway, thereby modulating various biological functions of gastric cancer cells and ultimately promoting the progression of gastric cancer. In summary, our findings suggest that IGF2BP3 upregulates the expression of FBXO32 protein in an m6A dependent manner and subsequently activates the cGMP-PKG signaling pathway, ultimately leading to the onset and progression of gastric cancer. Consequently, the targeting of the IGF2BP3/FBXO32/cGMP-PKG axis emerges as a promising therapeutic modality for the treatment of gastric cancer.

cancer biology↗

bioRxiv2024

Light regulates widespread plant alternative polyadenylation through the chloroplast

Transcription of eukaryotic protein-coding genes generates immature mRNAs that are subjected to a series of processing events, including capping, splicing, cleavage and polyadenylation (CPA) and chemical modifications of bases. Alternative polyadenylation (APA) greatly contributes to mRNA diversity in the cell. By determining the length of the 3 untranslated region, APA generates transcripts with different regulatory elements, such as miRNA and RBP binding sites, which can influence mRNA stability, turnover and translation. In the model plant Arabidopsis thaliana, APA is involved in the control of seed dormancy and flowering. In view of the physiological importance of APA in plants, we decided to investigate the effects of light/dark conditions and compare the underlying mechanisms to those elucidated for alternative splicing (AS). We found that light controls APA in approximately 30% of Arabidopsis genes. Similar to AS, the effect of light on APA requires functional chloroplasts, is not affected in mutants of the phytochrome and cryptochrome photoreceptor pathways and is observed in roots only when the communication with the photosynthetic tissues is not interrupted. Furthermore, mitochondrial activity is necessary for the effect of light in roots but not in shoots. However, unlike AS, coupling with transcriptional elongation does not seem to be involved since light-dependent APA regulation is neither abolished in mutants of the TFIIS transcript elongation factor nor universally affected by chromatin relaxation caused by the histone deacetylase inhibition. Instead, regulation seems to be linked to light-elicited changes in the abundance of constitutive CPA factors, also mediated by the chloroplast.

molecular biology↗

bioRxiv2024

Non-additive dosage-dependent effects of TaGS3 gene editing on grain size and weight in wheat

The grain size in cereals is one of the main component traits contributing to yield. Previous studies showed that loss-of-function (LOF) mutations in GS3, encoding G{gamma} subunit of the multimeric G protein complex, increase grain size and weight in rice. While association between allelic variation in GS3 homologs of wheat and grain weight/size was detected previusly, the effects of LOF alleles on these traits remain unknown. We used genome editing to create the TaGS3 mutant lines with the LOF homeo-allele dosage variation. Contrary to results obtained for rice, editing of all three TaGS3 copies result in significant decrease in grain length, width, grain area and weight, without affecting number of grains per spike. Compared to wild type, the highest increase in grain weight and area was observed in mutants with the intermediate dosage of the LOF alleles, indicating that suppressive effects of TaGS3 on grain size and weight in wheat are dosage-dependent and non-additive. Our results suggest that TaGS3 likely represents a functionally diverged homolog of GS3 evolved in the wheat lineage. The newly developed LOF alleles of TaGS3 expand the set of CRISPR-Cas9-induced variants of yield component genes that could be used for increasing grain weight in wheat.

genetics↗

bioRxiv2023

A molecularly distinct cell type in the dmPAG regulates intermale aggression behaviors in mice

BackgroundThe periaqueductal gray (PAG) is a central hub for regulation of aggression, while little is known on the circuitry and molecular mechanisms that govern this regulation. We investigate the role of a distinct cell type, Tachykinin 2-expressing (Tac2+) neurons, located in the dorsomedial PAG (dmPAG), in modulating aggression in mice. MethodsWe combined activity mapping, in vivo Ca2+ recording, chemogenetic and pharmacological manipulation, and a viral-based translating ribosome affinity purification (TRAP) profiling using a mouse resident-intruder model. ResultsWe reveal that the dmPAGTac2 neurons were selectively activated during fighting behaviors. Activation of the dmPAGTac2 neurons evoked, while inhibition or genetic ablation of the dmPAGTac2 neurons suppressed fighting behaviors. TRAP profiling of dmPAGTac2 neurons revealed that fighting behaviors specifically induced enrichment of serotonin-associated transcripts to the dmPAGTac2 neurons. Last, we validated these findings by selectively delivering pharmacological agent into the dmPAG and reversed the behavioral outcomes induced by chemogenetic manipulation. ConclusionsWe identify that the dmPAGTac2 neuron can regulate mouse aggressive behavior and thus suggest a distinct molecular target for the treatment of exacerbated aggressive behaviors in populations that exhibit high-level of violence. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/562724v3_ufig1.gif" ALT="Figure 1"> View larger version (75K): org.highwire.dtl.DTLVardef@afc876org.highwire.dtl.DTLVardef@91e355org.highwire.dtl.DTLVardef@1e41e43org.highwire.dtl.DTLVardef@17ef3f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Central pore-opening structure and gating of the mechanosensitive PIEZO1 channel

PIEZO1 is a mechanically activated cation channel that undergoes force-induced activation and inactivation to control diverse physiological processes1-4. However, the distinct functional states and gating transitions remain structurally undefined. In response to changes in membrane forces, PIEZO1 transitions from a curved to a flattened shape, which might lead to opening and subsequent inactivation of the ion-conducting pore5,6. Here we employed a PIEZO1 mutant to capture various conformational states including an intermediate state with an opened central pore, allowing us to reveal the gating dynamics of this multiple-gate system. Upon transition from the curved to the partially flattened intermediate state, the trimeric cap undergoes a downward and rotational motion to open the lateral cap-gate, while the loop that connects the cap to the pore-lining inner helix acts as a spring-linker, switching from an extended to a compressed state. Along with the partial flattening of the peripheral blades, these conformational changes collectively dilate the cap-gate, spring-gate and the hydrophobic transmembrane-gate, leading to ion permeation through the transmembrane pore. Upon inactivation, while the blades remain flattened, the cap and spring-linker return to an up and extended state. Molecular dynamics simulations verify the ion-permeating pathway and the distinct ion-conducting states of the assigned close, intermediate, open, and inactivated structures. Mutagenesis and electrophysiological characterizations reveal that domains and residues involved in conformational dynamics of the cap and spring-linker are critical for mechanical activation of PIEZO1. Together, these studies have structurally defined the distinct functional states and the curvature-based activation and inactivation mechanisms of PIEZO1.

neuroscience↗

bioRxiv2023

H3-OPT: Accurate prediction of CDR-H3 loop structures of antibodies with deep learning

Accurate prediction of the structurally diverse complementarity determining region heavy chain 3 (CDR-H3) loop structure remains a primary and long-standing challenge for antibody modeling. Here, we present the H3-OPT toolkit for predicting the 3D structures of monoclonal antibodies and nanobodies. H3-OPT combines the strengths of AlphaFold2 with a pre-trained protein language model, and provides a 2.24 [A] average RMSDC between predicted and experimentally determined CDR-H3 loops, thus outperforming other current computational methods in our non-redundant high-quality dataset. The model was validated by experimentally solving three structures of anti-VEGF nanobodies predicted by H3-OPT. We examined the potential applications of H3-OPT through analyzing antibody surface properties and antibody-antigen interactions. This structural prediction tool can be used to optimize antibody-antigen binding, and to engineer therapeutic antibodies with biophysical properties for specialized drug administration route.

bioinformatics↗

bioRxiv2022

Knowledge Mapping of Alternative Splicing of Cancer From 2012 to 2021: A Bibliometric Analysis

BackgroundAs a processing method of RNA precursors, alternative splicing plays a critical role in normal cell physiological activities. Aberrant alternative splicing events are associated with cancer development and are promising targets for cancer treatment. However, no detailed and unbiased study describes the current state of alternative splicing of cancer research. This study aims to conduct a bibliometric analysis of alternative splicing of cancer research in the last decade to measure and recognize the current state and trends. MethodsWeb of Science Core Collection was used to acquire the articles. Utilizing three bibliometric tools (CiteSpace, VOSviewer, R-bibliometrix), we were able to measure and recognize the influence and collaboration data of individual articles, journals, and co-citations. Analysis of co-occurrence and bursts information helped us identify the trending research areas related to alternative splicing of cancer. ResultsFrom 2012 to 2021, the total number of papers on alternative splicing of cancer published in 766 academic journals was 3,507, authored by 20,406 researchers in 405 institutions from 80 countries/regions. Research involving alternative splicing of cancer genes was primarily conducted in the United States and China, with the United States retaining the prime status; simultaneously, the Chinese Academy of Sciences, Fudan University and National Cancer Institute were the institutions with strong research capabilities. Scorilas Andreas is the scholar with the most publications, while the most co-citations were generated by Wang, Eric T. Plos One published the most papers on alternative splicing of cancer, while J biol chem was the most co-cited academic journal in this field. Furthermore, these publications covered a wide range of areas, but were still primarily focused on the fields of molecular, biology, and immunology. The results of keyword co-occurrence analysis can be divided into three types: molecular (P53, CD44, Androgen receptor, SRSF3, ESRP1), pathological process (apoptosis, EMT, metastasis, angiogenesis, proliferation), and disease (breast cancer, colorectal cancer, prostate cancer, hepatocellular carcinoma, gastric cancer). ConclusionResearch on alternative splicing of cancer has been a hot topic over the past decade, increasing in intensity each year. Current alternative splicing of cancer studies focused on hallmarks of alternative splicing in cancer, and alternative splicing signatures including diagnostic and therapeutic targets. Among them, the current trends are splicing factors regulating epithelial-mesenchymal transition and other hallmarks, aberrant splicing events in tumors, and further mechanisms. These might give researchers interested in this field a forward-looking perspective and inform further research.

cancer biology↗

bioRxiv2022

Wild-type allele of TaHRC suppresses calcium-mediated plant immune response by hijacking TaCAXIP4 to trigger FHB susceptibility in wheat

Fusarium head blight (FHB) is one of the most destructive diseases of wheat worldwide. Epidemics of FHB cause a serious reduction in grain yield and quality of wheat and result in significant economic losses to wheat producers. Recently, we have cloned a histidine-rich calcium-binding protein gene (TaHRC) as the causal gene for Fhb1 and demonstrated that the wild-type allele of TaHRC conditions FHB susceptibility and a large deletion including the start codon resulted in FHB resistance. However, the molecular mechanisms on how TaHRC regulating FHB susceptibility remains unknown. In this study, we conducted yeast two-hybrid screening (Y2H) against the wheat cDNA expression libraries using TaHRC as bait and identified a cation exchanger (CAX)-interacting protein 4 (TaCAXIP4) as the candidate protein that interacts with TaHRC to affect calcium transport activity. The strong interaction was further confirmed by Bimolecular fluorescence complementation (BiFC) assays. Using gene editing, we edited three different sites (one before and one within and one after the NLS domain) of TaHRC in a susceptible wheat cultivar Bobwhite using the CRISPR/Cas9 gene editing technology and demonstrated the N-terminus carrying NLS domain of TaHRC plays a critical role for the interaction and conditions TaHRC function on FHB susceptibility. We determined that the interaction between TaCAXIP4 and TaHRC occurs in the nuclei of cells by subcellular colocalization assay. Intriguingly, we found TaHRC can sequester TaCAXIP4 to suppress the Ca2+ transporting activity of TaCAX1 (a H+/Ca2+ antiporter) through yeast calcium suppression assay and suggested wild-type TaHRC may hijack TaCAXIP4 to suppresses calcium-mediated plant immune response during Fusarium infection in wheat. Furthermore, we performed the reactive oxygen species (ROS) assays and further showed that TaHRC might suppress the chitin-triggered plant immune responses during Fusarium infection by sequestering TaCAXIP4 to trigger FHB susceptibility, which facilitates the pathogen spread within a wheat spike. This work provides first line of evidence to support wild type Fhb1 is a susceptible gene and how Fhb1 wild type allele regulate FHB susceptibility.

plant biology↗

bioRxiv2022

Development and optimization of a Barley stripe mosaic virus (BSMV)-mediated gene editing system to improve Fusarium head blight (FHB) resistance in wheat

Fusarium head blight (FHB) is a devastating disease in wheat that causes million dollars of wheat yield losses annually in the U.S.A. Recently we demonstrated that wheat carry FHB susceptible genes and knocking out the susceptible gene increased FHB resistance, which suggests that manipulating susceptible genes using gene editing may open a new avenue to create new sources of FHB resistance. However, wheat genome editing uses gene transformation to deliver CRISPR/Cas9 and gRNA into plants, and most wheat genotypes are not suitable for transformation due to low rates of callus induction and regeneration, therefore it cannot be used in practical wheat breeding. In this study, we developed a new Barley stripe mosaic virus (BSMV)-mediated gRNA delivery system that does not need the gene transformation and tissue culture and it can be used in any genotypes for gene function validation and editing. We used this system edited the susceptible allele of Fhb1, a major FHB resistance gene. We demonstrated that the edited trait is heritable in different genetic backgrounds and knocking out Fhb1 susceptible allele improved FHB resistance in wheat. We also modified system to improve editing efficiency by using floral dip agroinfiltration and adding RNA mobility sequences to the gRNA in the viral vectors. We believe this work will facilitate wheat FHB resistance research and gene editing in cereal crops and will benefit cereal crop researchers and breeders worldwide.

plant biology↗

bioRxiv2021

HIF-1α stabilization modified by glutaredoxin-1 is critical for intestinal angiogenesis in NEC pathogenesis

BackgroundHypoxia inducible factor (HIF-1) are essential in the pathogenesis of necrotizing enterocolitis (NEC), which is stabilized by Grx1 deletion. Until now, the mechanism of HIF-1 in the intestinal microcirculation in NEC is not well defined. We intend to investigate the role of HIF-1 in the development of NEC in regulating the microcirculation and the following vasodilatory signal, VEGF. Materials and methodsExperimental NEC was induced in full-term C57BL/6 mouse and Grx1-/- pups through the formula gavage and hypoxia technique. The HIF-1 signal was blocked utilizing the HIF-1 inhibitor, YC-1. Intestinal tissues were collected at predetermined time points for the assessment of intestinal microcirculation and the HIF-1 activity involved signal. ResultsWe found that NEC inducement impaired the intestinal microcirculation, but intestinal blood flow and capillary density were ameliorated in Grx1-/- mice, which was associated with the GSH-protein adducts of HIF-1 in the intestinal tissue. Grx1 ablation could also promote vascular endothelial growth factor (VEGFA) production in the intestinal tissue. This intestinal microvascular improvement was not found in the HIF-1 inhibited mice, suggesting the HIF-1 dependent manner for intestinal microcirculatory perfusion. ConclusionThe current data demonstrated that HIF-1 signaling is involved in the intestinal microvascular modification during the pathogenesis of NEC, suggesting that targeting with HIF-1 might be a promising strategy for NEC treatment.

molecular biology↗

bioRxiv2021

Susceptibilities of human ACE2 genetic variants in coronavirus infection

The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in more than 1603 million cases worldwide and 3.4 million deaths (as of May 2021), with varying incidences and death rates among regions/ethnicities. Human genetic variation can affect disease progression and outcome, but little is known about genetic risk factors for SARS-CoV-2 infection. The coronaviruses SARS-CoV, SARS-CoV-2 and HCoV-NL63 all utilize the human protein angiotensin-converting enzyme 2 (ACE2) as the receptor to enter cells. We hypothesized that the genetic variability in ACE2 may contribute to the variable clinical outcomes of COVID-19. To test this hypothesis, we first conducted an in silico investigation of single-nucleotide polymorphisms (SNPs) in the coding region of ACE2 gene. We then applied an integrated approach of genetics, biochemistry and virology to explore the capacity of select ACE2 variants to bind coronavirus spike protein and mediate viral entry. We identified the ACE2 D355N variant that restricts the spike protein-ACE2 interaction and consequently limits infection both in vitro and in vivo. In conclusion, ACE2 polymorphisms could modulate susceptibility to SARS-CoV-2, which may lead to variable disease severity.

microbiology↗

bioRxiv2021

ZFC3H1 and U1-70K promote the nuclear retention of mRNAs with 5' splice site motif within nuclear speckles

Quality control of mRNA represents an important regulatory mechanism for gene expression in eukaryotes. One component of this quality control is the nuclear retention and decay of misprocessed RNAs. Previously, we demonstrated that mature mRNAs containing a 5 splice site (5SS) motif, which is typically found in misprocessed RNAs such as intronic polyadenylated (IPA) transcripts, are nuclear retained and degraded. Here we demonstrate that these transcripts require the zinc finger protein ZFC3H1 for their decay and nuclear retention into nuclear speckles. Furthermore, we find that U1-70K, a component of the U1 snRNP spliceosomal complex, is also required for their nuclear retention and likely functions in the same pathway as ZFC3H1. Finally, we show that the disassembly of nuclear speckles impairs the nuclear retention of mRNAs with 5SS motifs. Together, our results suggest a model where mRNAs with 5SS motifs are recognized by U1 snRNP, which then acts with ZFC3H1 to both promote their decay and prevent nuclear export of these mRNAs by sequestering them in nuclear speckles. Our results highlight a splicing independent role of U1 snRNP and indicate that it works in conjunction with ZFC3H1 in preventing the nuclear export of misprocessed mRNAs.

molecular biology↗

bioRxiv2021

CRISPRpas: Programmable regulation of alternative polyadenylation by dCas9

Well over half of human mRNA genes produce alternative polyadenylation (APA) isoforms that differ in mRNA metabolism due to 3 UTR size changes or have variable coding potentials when coupled with alternative splicing. Aberrant APA is implicated in a growing number of human diseases. A programmable tool for APA regulation, hence, would be instrumental for understanding how APA events impact biological processes. Here, using a catalytically dead Cas9 (dCas9), we developed a method, named CRISPRpas, to alter cleavage and polyadenylation site (PAS) usage in 3 UTRs or introns. We present key features that facilitate CRISPRpas, including targeting DNA strand, distance between PAS and targeting sequence, and strength of the PAS. For intronic PAS, we additionally analyze strengths of 5 splice site and target location in intron. Our analyses implicate a dynamic competition between PAS usage and nascent RNA decay when RNA polymerase II elongation is blocked. We show modulation of APA of multiple endogenous genes including a gene that contains a single nucleotide polymorphism (SNP) that affects APA in the human population. CRISPRpas expands the CRISPR toolkit for perturbation of gene expression.

molecular biology↗

bioRxiv2021

MAAPER: model-based analysis of alternative polyadenylation using 3' end-linked reads

Most eukaryotic genes harbor multiple cleavage and polyadenylation sites (PASs), leading to expression of alternative polyadenylation (APA) isoforms. APA regulation has been implicated in a diverse array of physiological and pathological conditions. While RNA sequencing tools that generate reads containing the PAS, named onSite reads, have been instrumental in identifying PASs, they have not been widely used. By contrast, a growing number of methods generate reads that are close to the PAS, named nearSite reads, including the 3 end counting strategy commonly used in single cell analysis. How these nearSite reads can be used for APA analysis, however, is poorly studied. Here, we present a computational method, named model-based analysis of alternative polyadenylation using 3 end-linked reads (MAAPER), to examine APA using nearSite reads. MAAPER uses a probabilistic model to predict PASs for nearSite reads with high accuracy and sensitivity, and examines different types of APA events, including those in 3UTRs and introns, with robust statistics. We show MAAPERs accuracy with data from both bulk and single cell RNA samples and its applicability in unpaired or paired experimental designs. Our result also highlights the importance of using well annotated PASs for nearSite read analysis.

bioinformatics↗

bioRxiv2021

SCAPTURE: a deep learning-embedded pipeline that captures polyadenylation information from 3' tag-based RNA-seq of single cells

Single-cell RNA-seq (scRNA-seq) profiles gene expression with a resolution that empowers depiction of cell atlas in complex systems. Here, we developed a stepwise computational pipeline SCAPTURE to identify, evaluate, and quantify cleavage and polyadenylation sites (PASs) from 3 tag-based scRNA-seq. SCAPTURE detects PASs de novo in single cells with high sensitivity and accuracy, enabling detection of previously unannotated PASs. Quantified alternative PAS transcripts refine cell identities, enriching information extracted from scRNA-seq.

bioinformatics↗

bioRxiv2021

Bacterium-Enabled Transient Gene Activation by Artificial Transcription Factor for Resolving Gene Regulation in Maize

Cellular functions are diversified through intricate transcription regulations, and an understanding gene regulation networks is essential to elucidating many developmental processes and environmental responses. Here, we employed the Transcriptional-Activator Like effectors (TALes), which represent a family of transcription factors that are synthesized by members of the {gamma}-proteobacterium genus Xanthomonas and secreted to host cells for activation of targeted host genes. Through delivery by the maize pathogen, Xanthomonas vasicola pv. vasculorum, designer TALes (dTALes), which are synthetic TALes, were used to induce the expression of the maize gene glossy3 (gl3), a MYB transcription factor gene involved in the cuticular wax biosynthesis. RNA-Seq analysis of leaf samples identified 146 gl3 downstream genes. Eight of the nine known genes known to be involved in the cuticular wax biosynthesis were up-regulated by at least one dTALe. A top-down Gaussian graphical model predicted that 68 gl3 downstream genes were directly regulated by GL3. A chemically induced mutant of the gene Zm00001d017418 from the gl3 downstream gene, encoding aldehyde dehydrogenase, exhibited a typical glossy leaf phenotype and reduced epicuticular waxes. The bacterial protein delivery of artificial transcription factors, dTALes, proved to be a straightforward and powerful approach for the revelation of gene regulation in plants.

plant biology↗

bioRxiv2021

Nanotraps for the containment and clearance of SARS-CoV-2

SARS-CoV-2 enters host cells through its viral spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptors on the host cells. Here we show functionalized nanoparticles, termed "Nanotraps", completely inhibited SARS-CoV-2 infection by blocking the interaction between the spike protein of SARS-CoV-2 and the ACE2 of host cells. The liposomal-based Nanotrap surfaces were functionalized with either recombinant ACE2 proteins or anti-SARS-CoV-2 neutralizing antibodies and phagocytosis-specific phosphatidylserines. The Nanotraps effectively captured SARS-CoV-2 and completely blocked SARS-CoV-2 infection to ACE2-expressing human cell lines and primary lung cells; the phosphatidylserine triggered subsequent phagocytosis of the virus-bound, biodegradable Nanotraps by macrophages, leading to the clearance of pseudotyped and authentic virus in vitro. Furthermore, the Nanotraps demonstrated excellent biosafety profile in vitro and in vivo. Finally, the Nanotraps inhibited pseudotyped SARS-CoV-2 infection in live human lungs in an ex vivo lung perfusion system. In summary, Nanotraps represent a new nanomedicine for the inhibition of SARS-CoV-2 infection. HighlightsO_LINanotraps block interaction between SARS-CoV-2 spike protein and host ACE2 receptors C_LIO_LINanotraps trigger macrophages to engulf and clear virus without becoming infected C_LIO_LINanotraps showed excellent biosafety profiles in vitro and in vivo C_LIO_LINanotraps blocked infection to living human lungs in ex vivo lung perfusion system C_LI Progress and PotentialTo address the global challenge of creating treatments for SARS-CoV-2 infection, we devised a nanomedicine termed "Nanotraps" that can completely capture and eliminate the SARS-CoV-2 virus. The Nanotraps integrate protein engineering, immunology, and nanotechnology and are effective, biocompatible, safe, stable, feasible for mass production. The Nanotraps have the potential to be formulated into a nasal spray or inhaler for easy administration and direct delivery to the respiratory system, or as an oral or ocular liquid, or subcutaneous, intramuscular or intravenous injection to target different sites of SARS-CoV-2 exposure, thus offering flexibility in administration and treatment. More broadly, the highly versatile Nanotrap platform could be further developed into new vaccines and therapeutics against a broad range of diseases in infection, autoimmunity and cancer, by incorporating with different small molecule drugs, RNA, DNA, peptides, recombinant proteins, and antibodies.

bioengineering↗

bioRxiv2020

Expanding the range of editable targets in the wheat genome using the variants of the Cas12a and Cas9 nucleases

The development of CRISPR-based editors having different protospacer adjacent motif (PAM) recognition specificities, or guide RNA length/structure requirements broadens the range of possible genome editing applications. Here, we evaluated the natural and engineered variants of Cas12a (FnCas12a from Francisella novicida and LbCas12a from Lachnospiraceae bacterium) and Cas9 for wheat genome editing efficiency and ability to induce heritable mutations in endogenous genes controlling important agronomic traits in wheat. Unlike FnCas12a, LbCas12a was able to induce mutations in the wheat genome in the current study, even though with a lower rate than that reported for SpCas9. The eight-fold improvement in the gene editing efficiency was achieved for LbCas12a by using the guide RNAs flanked by ribozymes and driven by the RNA polymerase II promoter from switchgrass. The efficiency of multiplexed genome editing (MGE) using LbCas12a was mostly similar to that obtained using the simplex RNA guides. A LbCas12a-MGE construct was successfully applied for generating heritable mutations in a gene controlling grain size and weight in wheat. We show that the range of editable loci in the wheat genome could be expanded by using the engineered variants of Cas12a (LbCas12a-RVR) and Cas9 (Cas9-NG and xCas9) that recognize the TATV and NG PAMs, respectively, with the Cas9-NG showing higher editing efficiency on the targets with atypical PAMs compared to xCas9. In conclusion, our study reports the set of validated natural and engineered variants of Cas12a and Cas9 editors for targeting loci in the wheat genome not amenable to Cas9-based modification.

genetics↗