Tag: HEK293T

Thyroid hormone-regulated chromatin landscape and transcriptional sensitivity of the pituitary gland

Mouse genetic models The ThrbHAB allele expresses TRβ proteins (TRβ1 and TRβ2) fused to a peptide with a hemagglutinin (HAx2) tag and a site for biotinylation by prokaryotic BirA ligase, modified from a published tag30. The tag was inserted at the endogenous Thrb gene by homologous recombination in W9.5 (129/Sv)…

Continue Reading Thyroid hormone-regulated chromatin landscape and transcriptional sensitivity of the pituitary gland

Employment of a high throughput functional assay to define the critical factors that influence vaccine induced cross-variant neutralizing antibodies for SARS-CoV-2

Study design, ethical statement, and sample collection The vaccinated participants were recruited under the COVID-19 PROTECT study (2012/00917). The convalescent plasma samples were collected from subjects who were diagnosed with COVID-19 by positive polymerase chain reaction (PCR) results (study 2020/00120). All participants provided written informed consent in accordance with the…

Continue Reading Employment of a high throughput functional assay to define the critical factors that influence vaccine induced cross-variant neutralizing antibodies for SARS-CoV-2

Additional file 1 of DNA polymerases in precise and predictable CRISPR/Cas9-mediated chromosomal rearrangements

journal contribution posted on 2023-12-09, 04:41 authored by Mohammadreza M. Mehryar, Xin Shi, Jingwei Li, Qiang Wu Additional file 1: Fig. S1. DNA-fragment editing by Cas9 with dual sgRNAs. Fig. S2. HPRT1 and DCK reporter assay systems for Cas9-induced large resections. Fig. S3. Quantitative RT-PCR at two time points in HEC-1-B…

Continue Reading Additional file 1 of DNA polymerases in precise and predictable CRISPR/Cas9-mediated chromosomal rearrangements

DNA polymerases in precise and predictable CRISPR/Cas9-mediated chromosomal rearrangements | BMC Biology

Cell culture The human endometrial carcinoma HEC-1-B cells were cultured in the modified Eagle’s medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a 5% (v/v) CO2 incubator. The human embryonic kidney HEK293T cells were cultured in the Dulbecco’s modified Eagle’s medium (DMEM) supplemented…

Continue Reading DNA polymerases in precise and predictable CRISPR/Cas9-mediated chromosomal rearrangements | BMC Biology

High-throughput ligand profile characterization in novel cell lines expressing seven heterologous insect olfactory receptors for the detection of volatile plant biomarkers

Generation of the stable OR cell lines In vivo measured ligand profile data derived from the DoOR database (neuro.uni-konstanz.de/DoOR/default.html)19,20 were used to select 12 ORs, which can be appropriate to detect the pathogen-related VOCs relevant for our study. To assemble a novel cell-based olfactory panel, 11 stable cell lines were…

Continue Reading High-throughput ligand profile characterization in novel cell lines expressing seven heterologous insect olfactory receptors for the detection of volatile plant biomarkers

tRNA therapeutics for genetic diseases

Huang, X. et al. The landscape of mRNA nanomedicine. Nat. Med. 28, 2273–2287 (2022). Article  CAS  Google Scholar  Rohner, E., Yang, R., Foo, K. S., Goedel, A. & Chien, K. R. Unlocking the promise of mRNA therapeutics. Nat. Biotechnol. 40, 1586–1600 (2022). Article  CAS  Google Scholar  Brown, A., Shao, S.,…

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Precise base editing without unintended indels in human cells and mouse primary myoblasts

Base editors cause unintended indels at the target sites Several types of evolved base editors based on the CRISPR system have been developed for more accurate and efficient genome engineering21,37. Among these, AncBE4max and ABEmax were evolved by modifying codon usage, NLSs, and ancestral deaminase reconstructions21. These modifications greatly improve…

Continue Reading Precise base editing without unintended indels in human cells and mouse primary myoblasts

High-throughput screening of genetic and cellular drivers of syncytium formation induced by the spike protein of SARS-CoV-2

Plasmid construction All the constructs used in this study were generated with standard cloning strategies, including PCR, overlapping PCR, oligo annealing, digestion and ligation. Primers were purchased from Genewiz. The plasmid sequence was verified by Sanger sequencing. The pCAG-spike(D614G)-GFP11-mCherry plasmid was modified from Addgene plasmid 158761. Briefly, GFP11 and mCherry…

Continue Reading High-throughput screening of genetic and cellular drivers of syncytium formation induced by the spike protein of SARS-CoV-2

LncRNA INHEG promotes glioma stem cell maintenance and tumorigenicity through regulating rRNA 2’-O-methylation

Ethics statement All mice procedures in this study were performed under an animal protocol approved by the Institutional Animal Care and Use Committee guidelines of Westlake University. The procedures and protocols for glioma patients were approved by the institutional review board of Beijing Tiantan Hospital. Informed consent was obtained from…

Continue Reading LncRNA INHEG promotes glioma stem cell maintenance and tumorigenicity through regulating rRNA 2’-O-methylation

FAT10 is phosphorylated by IKK{beta} to inhibit the antiviral type-I interferon response

Introduction The innate immune system represents the host´s first-line defense against viral infections (Koyama et al, 2008). In this context, IFN-I are produced by all nucleated cells and represent the principal cytokines that counteract viral replication (Ivashkiv & Donlin, 2014). In fact, several receptors of the infected cell can recognize…

Continue Reading FAT10 is phosphorylated by IKK{beta} to inhibit the antiviral type-I interferon response

Both chimpanzee adenovirus-vectored and DNA vaccines induced long-term immunity against Nipah virus infection

Ethics statement The animal experiments were approved by the Animal Ethics Committee of the Wuhan Institute of Virology, Chinese Academy of Sciences (approval number: WIVA21202104). All animal experiments involving NiV were executed in the animal biosafety level 4 (ABSL-4) facility at the National Biosafety Laboratory (Wuhan), Chinese Academy of Sciences….

Continue Reading Both chimpanzee adenovirus-vectored and DNA vaccines induced long-term immunity against Nipah virus infection

CRISPR screening in hematology research: from bulk to single-cell level | Journal of Hematology & Oncology

Transcriptomics CROP-seq [46] Jurkat Poly-A CRISPRko Cas9 119 – 5798 (+ 1320 cells with NT gRNA) Lentiviral DROP-seq RNA Perturb-seq [43, 44] K562 Barcode CRISPRi dCas9-KRAB UPR epistasis screen: 9 triplet combinations UPR Perturb-seq experiment: 91 Up to 3 gRNAs in a single vector UPR epistasis screen: 15006 UPR Perturb-seq experiment:…

Continue Reading CRISPR screening in hematology research: from bulk to single-cell level | Journal of Hematology & Oncology

TET2 modulates spatial relocalization of heterochromatin in aged hematopoietic stem and progenitor cells

Animal models Animal studies were approved by the Institutional Animal Care Use Committee of the Institute of Biosciences and Technology, Texas A&M University (AUP 2020-0195). Most mouse strains bear a C57BL/6 genetic background unless otherwise noted. All the animals were housed in a certified animal facility with a standard dark/light…

Continue Reading TET2 modulates spatial relocalization of heterochromatin in aged hematopoietic stem and progenitor cells

Inactive S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in human stem cell model of Parkinson’s disease

Cloning of CRISPR/sgRNA lentiviral constructs with fluorescent selection markers A tetracycline-inducible promoter (TRE3G) was used to control the expression of S. aureus dCas9 in a lentiviral vector. To facilitate selection of cells by FACS, pHR:TRE3G-SadCas9-2xKRAB-p2a-tdTomato (Addgene ID #209298) was subcloned from a pHR:TRE3G-SadCas9-2xKRAB-p2a-zeo (A gift from Professor Stanley Qi), where zeocin…

Continue Reading Inactive S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in human stem cell model of Parkinson’s disease

Generation of UL128-shRNA transduced fibroblasts for the release of cell-free virus from clinical human cytomegalovirus isolates

Human cytomegalovirus (HCMV) is a herpesvirus that is widespread in the population worldwide [1,2], where it remains latent after infection in its host for a lifetime [3–5]. While primary infection or reactivation in immunocompetent individuals usually remains subclinical and causes little or no symptoms, it can cause severe damage in…

Continue Reading Generation of UL128-shRNA transduced fibroblasts for the release of cell-free virus from clinical human cytomegalovirus isolates

Mitigating a TDP-43 proteinopathy by concentrating on ataxin-2 utilizing

Concentrating on ataxin-2 with RfxCas13d Due to its potential to affect TDP-43-associated toxicity20,25,26, ataxin-2 has emerged as a doubtlessly broadly relevant goal for ALS-FTD, as TDP-43 pathology is noticed in ~97% of ALS instances1,4,5 and ~45% FTD occurrences1,2,4. Given this, we sought to find out if RNA-targeting CRISPR effector proteins…

Continue Reading Mitigating a TDP-43 proteinopathy by concentrating on ataxin-2 utilizing

DNA damage induced by CDK4 and CDK6 blockade triggers anti-tumor immune responses through cGAS-STING pathway

Patients’ tumor tissues and clinical data This study recruited 125 breast cancer patients for immunohistochemical (IHC) analysis and 10 breast cancer patients for RNA-seq analysis from the Affiliated Tumor Hospital of Nantong University. Tumor tissues collected from surgical patients were fixed with formalin and embedded with paraffin and then examined….

Continue Reading DNA damage induced by CDK4 and CDK6 blockade triggers anti-tumor immune responses through cGAS-STING pathway

Apoptotic stress causes mtDNA release during senescence and drives the SASP

Cell culture and treatments Human embryonic lung MRC5 fibroblasts (ATCC) and IMR90 fibroblasts (ATCC) were grown in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, D5796) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U ml−1 penicillin, 100 μg ml−1 streptomycin and 2 mM l-glutamine and maintained at 37 °C under 5% CO2. MRC5 fibroblasts were cultured in…

Continue Reading Apoptotic stress causes mtDNA release during senescence and drives the SASP

Rab41-mediated ESCRT machinery repairs membrane rupture by a bacterial toxin in xenophagy

Rab8A and Rab41 are required for xenophagy of GAS For comprehensive screening of Rab GTPases involved in xenophagy against GAS infection, we first knocked down the expression of Rab GTPases in HeLa cells. We examined bacterial survival in cells at 6 h post-infection (hpi) compared with that at 2 hpi because…

Continue Reading Rab41-mediated ESCRT machinery repairs membrane rupture by a bacterial toxin in xenophagy

In planta expression of human polyQ-expanded huntingtin fragment reveals mechanisms to prevent disease-related protein aggregation

Plant material and constructs A. thaliana lines of Columbia-0 (Col-0) ecotype were employed, including WT, toc159 (refs. 58,59) and cct8-2 (ref. 60). Seeds underwent surface sterilization and germination on solid 0.5× Murashige and Skoog (M&S) medium with vitamins, lacking sucrose. Plants were incubated in a growth chamber at 22 °C under…

Continue Reading In planta expression of human polyQ-expanded huntingtin fragment reveals mechanisms to prevent disease-related protein aggregation

TRIM25 targets p300 for degradation

Introduction Protein levels are regulated at several nodes. One mode of protein level regulation acts through enhancing or reducing gene transcription. Gene transcription can be stimulated by binding transcription factors to promoters or enhancers of target genes and by posttranslational modifications of transcription factors and histones. Such modifications can be…

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IJMS | Free Full-Text | CRISPR-Cas9 Direct Fusions for Improved Genome Editing via Enhanced Homologous Recombination

Over the past decade, CRISPR-Cas9 has found widespread application in loss-of-function mutations, but precise genetic engineering for gene correction or gene replacement therapies has lagged behind. In vivo correction using CRISPR-Cas9 to replace genetic mutations by HR is highly challenging, and very few studies have managed to achieve this [31,32]….

Continue Reading IJMS | Free Full-Text | CRISPR-Cas9 Direct Fusions for Improved Genome Editing via Enhanced Homologous Recombination

Specific Modulation of CRISPR Transcriptional Activators through RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos

Modular iSBH-sgRNA designs enable spatial separation of spacer and trigger-sensing sequences. A. In second-generation iSBH-sgRNAs, RNA triggers are complementary with the iSBH-sgRNA backfolds, thus sgRNA spacers influence RNA trigger sequences. In modular iSBH-sgRNAs, design constrains were eliminated as triggers are only complementary with the iSBH-sgRNA loop and first 15nt of…

Continue Reading Specific Modulation of CRISPR Transcriptional Activators through RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos

ncRNA | Free Full-Text | The Intergenic Type lncRNA (LINC RNA) Faces in Cancer with In Silico Scope and a Directed Lens to LINC00511: A Step toward ncRNA Precision

CRC Upregulated 153-5p HIF-1 activates LINC00511, targeting HIF-1’s 3-UTR and +ve feedback loop [78] Oncogene Human CRC tissues vs. paired adjacent non-tumor and CRC cell lines HT29, LOVO, SW620, SW480 vs. normal colon epithelial FHC cell line and athymic BALB/c nude mice for implantation – HNF4 promotes LINC00511 transcription, interaction…

Continue Reading ncRNA | Free Full-Text | The Intergenic Type lncRNA (LINC RNA) Faces in Cancer with In Silico Scope and a Directed Lens to LINC00511: A Step toward ncRNA Precision

A computationally designed antigen eliciting broad humoral responses against SARS-CoV-2 and related sarbecoviruses

Phylogenetic analysis Protein sequences of spike proteins were downloaded from the NCBI virus database for all the known sarbecoviruses (June 2020). A multiple sequence alignment was generated using MUSCLE36. The resulting multiple sequence alignment was pruned to the RBD region, filtered at 95% sequence identity and used as input for…

Continue Reading A computationally designed antigen eliciting broad humoral responses against SARS-CoV-2 and related sarbecoviruses

Split gene-editing tool offers greater precision

Chemically inducible split ABE (sABE) with tightly regulated deaminase activity. a Schematic of the EYFP fluorescence. An A-to-G conversion on the highlighted adenine on the antisense strand of the dysfunctional EYFP* gene can restore the expression of functional EYFP protein. b Schematic of the sABE. In the absence of rapamycin…

Continue Reading Split gene-editing tool offers greater precision

Transcriptional linkage analysis with in vivo AAV-Perturb-seq

Experimental procedures Plasmid design and cloning AAV genome plasmids (Fig. 1a and Extended Data Figs. 1a,g,h and 5a) were based on Addgene plasmid 60231 (ref. 12). To achieve widespread transgene expression, the hSyn promoter was replaced by the ubiquitous CBh promoter (pAS088). For the triple-colour experiments (Extended Data Fig. 1a),…

Continue Reading Transcriptional linkage analysis with in vivo AAV-Perturb-seq

CCDC50, an essential driver involved in tumorigenesis, is a potential severity marker of diffuse large B cell lymphoma

Data collection and bioinformatic analysis GSE10846, GSE19246, GSE32918, GSE50721, GSE64820, and GSE94669, were downloaded from the Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo/). DLBCL datasets from the GEPIA (Gene Expression Profiling Interactive Analysis) (gepia.cancer-pku.cn) and the TCGA (The Cancer Genome Atlas) (xenabrowser.net/datapages/) were also used in this study. A total of 284…

Continue Reading CCDC50, an essential driver involved in tumorigenesis, is a potential severity marker of diffuse large B cell lymphoma

Scientists Develop Novel Base Editing System

Register for free to listen to this article Thank you. Listen to this article using the player above. ✖ Want to listen to this article for FREE? Complete the form below to unlock access to ALL audio articles. In a study published in Nature Biotechnology on Aug. 28, GAO…

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Novel based editors developed – Drug Target Review

Researchers have developed a novel based CRISPR-free base editing system named CyDENT, which enables precise base editing. In a study published in Nature Biotechnology, GAO Caixia’s team at the Institute of Genetics and Developmental Biology (IGDB) of the Chinese Academy of Sciences, in collaboration with researchers at Qi Biodesign, has introduced…

Continue Reading Novel based editors developed – Drug Target Review

Base Editors Show Strand-Specific Targeting in Nuclei and Organelles

Researchers from the Chinese Academy of Scientists (CAS) and their collaborators at Qi Biodesign have developed a strand-specific, CRISPR-free editing system for modifying genomes in the nucleus, mitochondria, and chloroplasts of plant and human cells. The team was led by Caixia Gao, PhD, a principal investigator at the Institute of…

Continue Reading Base Editors Show Strand-Specific Targeting in Nuclei and Organelles

Dual CRISPR-Cas3 is a promising tool to induce a gigantic genomic deletion and restore dystrophin protein

In a recent study published in Stem Cell Reports, researchers evaluated the use of a dual clustered regularly interspaced short palindromic repeats (CRISPR)-Cas3 system for inducing multi-exon skipping (MES) among Duchenne muscular dystrophy (DMD)-patient-derived induced pluripotent stem cells (iPSCs). Study: Dual CRISPR-Cas3 system for inducing multi-exon skipping in DMD patient-derived iPSCs. Image…

Continue Reading Dual CRISPR-Cas3 is a promising tool to induce a gigantic genomic deletion and restore dystrophin protein

CRISPR Approach to Induce Exon Skipping in Dystrophin Gene

A dual CRISPR-Cas3 system could be used to induce multi-exon skipping to restore the open reading frame of the dystrophin gene, a new study has found. Duchenne muscular dystrophy, a severe degenerative muscle disorder, is caused by mutations in the dystrophin gene that disrupt its open reading frame, leading to…

Continue Reading CRISPR Approach to Induce Exon Skipping in Dystrophin Gene

AAV-mediated base-editing therapy ameliorates the disease phenotypes in a mouse model of retinitis pigmentosa

Ethics statement This study did not involve human data. All animal procedures were approved by the Institutional Review Board of Shanghai Jiao Tong University and were conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Visual Research. Animals…

Continue Reading AAV-mediated base-editing therapy ameliorates the disease phenotypes in a mouse model of retinitis pigmentosa

Optimized bisulfite sequencing analysis reveals the lack of 5-methylcytosine in mammalian mitochondrial DNA

doi: 10.1186/s12864-023-09541-9. Affiliations Expand Affiliations 1 State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, 200031, China. shaozhenyu2017@sibcb.ac.cn. 2 Shanghai Key Laboratory of Medical Epigenetics, Institutes of Biomedical…

Continue Reading Optimized bisulfite sequencing analysis reveals the lack of 5-methylcytosine in mammalian mitochondrial DNA

CRISPR library screening to develop HEK293-derived cell lines with improved lentiviral vector titers

doi: 10.3389/fgeed.2023.1218328. eCollection 2023. Affiliations Expand Affiliation 1 Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United States. Free PMC article Item in Clipboard Brian J Iaffaldano et al. Front Genome Ed. 2023. Free PMC article Show details Display…

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Genes | Free Full-Text | Combined Single Gene Testing and Genome Sequencing as an Effective Diagnostic Approach for Anophthalmia and Microphthalmia Patients

4. Discussion A/M is a group of structural ocular defects with varying degrees of severity, ranging from unilateral to bilateral and from simplex (non-syndromic) to complex (syndromic) types. A/M is a complicated disorder, and the underlying mechanisms of the disease are still poorly understood. Some suggest that A/M disorders arise…

Continue Reading Genes | Free Full-Text | Combined Single Gene Testing and Genome Sequencing as an Effective Diagnostic Approach for Anophthalmia and Microphthalmia Patients

Zfp296 knockout enhances chromatin accessibility and induces a unique state of pluripotency in embryonic stem cells

Establishment of Zfp296-deficient ESCs The expression of Zfp296 during ESC differentiation into trophectoderm was examined using the ZHBTc4 ESC line, in which expression of Pou5f1 encoding Oct3/4 can be downregulated by tetracycline20. As shown in Supplementary Fig. 1a, Zfp296 expression rapidly decreased, suggesting that Zfp296 is downstream of Oct3/4. To generate…

Continue Reading Zfp296 knockout enhances chromatin accessibility and induces a unique state of pluripotency in embryonic stem cells

Phenotypic and molecular basis of SIX1 variants linked to non-syndromic deafness and atypical branchio-otic syndrome in South Korea

Clinical phenotypes This study included eight affected patients (three males and five females) from five unrelated families, segregating with SIX1 variants (Fig. 1a). A comprehensive overview of their clinical phenotypes, encompassing both major and minor criteria, is provided in Table 1. All eight individuals (100%) experienced sensorineural hearing loss (SNHL), with…

Continue Reading Phenotypic and molecular basis of SIX1 variants linked to non-syndromic deafness and atypical branchio-otic syndrome in South Korea

Amphioxus adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U and A-to-I deamination of DNA

Animals and cells Adult amphioxus B. japonicum collected from the sandy bottom of the sea near Qingdao, China, were cultured in aerated seawater at room temperature and fed with single-celled algae once a day. Zebrafish (D. rerio) aged 3 months and adult Japanese scallops (M. yessoensis) were purchased from Nanshan…

Continue Reading Amphioxus adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U and A-to-I deamination of DNA

In vitro and in vivo characterization of SARS-CoV-2 resistance to ensitrelvir

Ethics All animal experiments were conducted in accordance with the University of Tokyo’s Regulations for Animal Care and Use, which were approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo. The committee acknowledged and accepted both the legal and ethical responsibility for the…

Continue Reading In vitro and in vivo characterization of SARS-CoV-2 resistance to ensitrelvir

The combined action of the intracellular regions regulates FGFR2 kinase activity

To provide structure and clarity to the understanding of the intricate control mechanism herein investigated, we have deconstructed intracellular FGFR2 to the three principle regulatory regions: JM, KD and CT. This provides an understanding of how both JM and CT influence KD activation and dimerization. Through subsequent reconstruction of their…

Continue Reading The combined action of the intracellular regions regulates FGFR2 kinase activity

HIV-1 promotes ubiquitination of the amyloidogenic C-terminal fragment of APP to support viral replication

Ethics Statement LifeSource Buffy Coat blood obtained from New York blood center was used for isolation of peripheral blood mononuclear cells using Ficoll-Plaque (GE). Ethical approval for the study was obtained from the Institutional Review Board of Northwestern University and all donors provided their written, informed consent. Cell cultures HEK293T…

Continue Reading HIV-1 promotes ubiquitination of the amyloidogenic C-terminal fragment of APP to support viral replication

Reduced Vrk2 expression is associated with higher risk of depression in humans and mediates depressive-like behaviors in mice | BMC Medicine

The strategy and logic of choosing VRK2 SNPs for genetic analyses We retrieved the summary statistics of VRK2 SNPs in 23andMe, UK Biobank, and PGC2 samples [2, 3], and 256 SNPs region were available in all three GWAS datasets (a total of 246,363 cases and 561,190 controls). An overview about…

Continue Reading Reduced Vrk2 expression is associated with higher risk of depression in humans and mediates depressive-like behaviors in mice | BMC Medicine

A framework for individualized splice-switching oligonucleotide therapy

Patients The WGS and clinical data of 235 patients with A-T were provided by the Global A-T Family Data Platform of ATCP. Our access to the data was approved by the Data Access Committee of ATCP. Selected patients with A-T enrolled at the Manton Center for Orphan Disease Research under…

Continue Reading A framework for individualized splice-switching oligonucleotide therapy

Generation of hiPSCs with ABO c.767T>C substitution: resulting in splicing variants

. 2023 Jun 15;14:1141756. doi: 10.3389/fgene.2023.1141756. eCollection 2023. Yinge Jin  1 , Tao Chen  1 , Wei Zheng  1 , Jiahui Xi  1 , Yin Zi  1 , Jinling Wang  1 , Yue Chi  1 , Min Chen  1 , Qingjian Zou  1 , Chengcheng Tang  1 , Liangxue Lai  1   2 , Xiaoqing Zhou  1 Affiliations Expand Affiliations 1…

Continue Reading Generation of hiPSCs with ABO c.767T>C substitution: resulting in splicing variants

CRISPR/Cas9-based screening identifies potential therapies for non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is characterized by an excessive accumulation of lipids in hepatocytes in non-alcoholic individuals. NAFLD progressively changes from fatty liver to non-alcoholic steatohepatitis (NASH), cirrhosis, fibrosis, and, in some cases, hepatocellular carcinoma. Since NAFLD significantly affects morbidity, healthcare services, and quality of life, it is imperative…

Continue Reading CRISPR/Cas9-based screening identifies potential therapies for non-alcoholic fatty liver disease

CRISPR prime editing for unconstrained correction of oncogenic KRAS variants

Design of universal pegRNAs for KRAS gene correction We first investigated the mutation frequency of three RAS family genes, HRAS, NRAS, and KRAS, which are involved in tumorigenesis. According to the COSMIC (Catalog Of Somatic Mutations In Cancer) database (cancer.sanger.ac.uk/)22, KRAS mutations account for the majority (81.4%) of RAS mutations,…

Continue Reading CRISPR prime editing for unconstrained correction of oncogenic KRAS variants

CAR T cells as micropharmacies against solid cancers: Combining effector T-cell mediated cell death with vascular targeting in a one-step engineering process

General Lab Operation The assays were performed by experienced individuals throughout the course of the study. The study was performed using established laboratory protocols covering the processing, freezing, storage and thawing of cells as well as the staining procedure, data acquisition and gating strategy. Additional data beyond the data found…

Continue Reading CAR T cells as micropharmacies against solid cancers: Combining effector T-cell mediated cell death with vascular targeting in a one-step engineering process

SMCHD1 and LRIF1 converge at the FSHD-associated D4Z4 repeat and LRIF1 promoter yet display different modes of action

Somatic loss of LRIF1 or SMCHD1 in control myocytes leads to insufficient DUX4 derepression We have previously shown that SMCHD1 and LRIF1L aid in transcriptional repression of the D4Z4 repeat as short-term depletion of either LRIF1L or SMCHD1 in muscle cells having a D4Z4 repeat of <20 units on a…

Continue Reading SMCHD1 and LRIF1 converge at the FSHD-associated D4Z4 repeat and LRIF1 promoter yet display different modes of action

Genomic screening of 16 UK native bat species through conservationist networks uncovers coronaviruses with zoonotic potential

Sample collection Sampling kits were sent out to various bat rehabilitators in the UK, as described previously56, for the collection of faeces from bats. These faecal samples (0.02–1 g) were immediately stored in 5 ml of RNAlater solution to prevent degradation of RNA. The geographical locations and collection dates for all samples…

Continue Reading Genomic screening of 16 UK native bat species through conservationist networks uncovers coronaviruses with zoonotic potential

Engineered circular guide RNAs boost CRISPR/Cas12a- and CRISPR/Cas13d-based DNA and RNA editing | Genome Biology

Plasmid construction For U6+27 gRNA-expressing constructs, the DNA sequences including the hU6+27 promoter, a gRNA scaffold, and a gRNA insert site were synthesized and cloned into the pBluSKM vector. For Pre gRNA-expressing constructs, a second gRNA scaffold was inserted downstream of the gRNA insert site in the U6+27 gRNA-expressing constructs….

Continue Reading Engineered circular guide RNAs boost CRISPR/Cas12a- and CRISPR/Cas13d-based DNA and RNA editing | Genome Biology

Development of Specialized Microelectrode Arrays with Local Electroporation Functionality

Fabrication and Encapsulation of MEAs Borosilicate glass wafers with 100 mm (4ʺ) diameter were used as substrates for the MEA structures. For a lift-off process (Fig. 2a-1 and a-2), AZ5215E photoresist (MicroChemicals GmbH, Ulm, Germany) was spin-coated and patterned by an image reversal UV photolithography step. Then, 10 nm titanium was deposited…

Continue Reading Development of Specialized Microelectrode Arrays with Local Electroporation Functionality

Design of bacteriophage T4-based artificial viral vectors for human genome remodeling

Assembly of T4 artificial viral vectors T4-AVVs were assembled by sequential incorporation of purified biomaterials to generate a virus structural mimic (Fig. 2a and Supplementary Movie 1). Starting with an empty capsid shell purified from E. coli infected by the neck-minus and tail-minus T4 phage mutant (10-amber.13-amber.HocΔ.SocΔ T4) (Supplementary Fig. 1a), a pentameric…

Continue Reading Design of bacteriophage T4-based artificial viral vectors for human genome remodeling

A previously uncharacterized Factor Associated with Metabolism and Energy (FAME/C14orf105/CCDC198/1700011H14Rik) is related to evolutionary adaptation, energy balance, and kidney physiology

Statement on ethical considerations All animal work was approved and permitted by the Local Ethical Committee on Animal Experiments and conducted according to the Guidelines for Animal Experimentation recommendations (ARRIVE guidelines). In particular, mouse work related to C57BL/6NCrl mice was approved and permitted by the Institute of Molecular Genetics of…

Continue Reading A previously uncharacterized Factor Associated with Metabolism and Energy (FAME/C14orf105/CCDC198/1700011H14Rik) is related to evolutionary adaptation, energy balance, and kidney physiology

Clinical characteristics and host immunity responses of SARS-CoV-2 Omicron variant BA.2 with deletion of ORF7a, ORF7b and ORF8 | Virology Journal

Study cohorts This study was conducted in compliance with the Declaration of Helsinki and was approved by the Maoming People’s Hospital (Approval No. 2021-hs-43) and Dongguan Ninth People’s Hospital (Approval No. 2022-8). COVID-19 was confirmed based on positive results of reverse transcription-polymerase chain reaction (RT-PCR) of nasopharyngeal samples. Whole blood…

Continue Reading Clinical characteristics and host immunity responses of SARS-CoV-2 Omicron variant BA.2 with deletion of ORF7a, ORF7b and ORF8 | Virology Journal

Vaccinia E5 is a major inhibitor of the DNA sensor cGAS

Screening strategy for identifying viral inhibitors of the cGAS/STING pathway Vaccinia virus is a large cytoplasmic DNA virus with a 190 kilobase pairs (kbp) genome that encodes over 200 proteins21,22. MVA has an approximately 30-kbp deletion from its parental vaccinia genome, resulting in the loss of many immune-modulatory viral genes15….

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Novel ‘CLEVER’ method accelerates engineering and genetic study of SARS-CoV-2 and its variants

A recent study posted to the bioRxiv*preprint server described rapid mutagenesis and rescue of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants without cloning, using a novel reverse genetics strategy. Study: Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform. Image Credit: kentoh/Shutterstock.com *Important notice: bioRxiv publishes preliminary…

Continue Reading Novel ‘CLEVER’ method accelerates engineering and genetic study of SARS-CoV-2 and its variants

Convergent evolution of SARS-CoV-2 Omicron subvariants leading to the emergence of BQ.1.1 variant

Ethics statement All experiments with hamsters were performed in accordance with the Science Council of Japan’s Guidelines for the Proper Conduct of Animal Experiments. The protocols were approved by the Institutional Animal Care and Use Committee of National University Corporation Hokkaido University (approval ID: 20-0123 and 20-0060). All protocols involving…

Continue Reading Convergent evolution of SARS-CoV-2 Omicron subvariants leading to the emergence of BQ.1.1 variant

Optimized metrics for orthogonal combinatorial CRISPR screens

Plasmid design The information on each plasmid for combinatorial libraries is indicated in Supplementary Table S1. Plasmids used in this study to generate combinatorial libraries for SpCas9, enAsCas12a, CHyMErA, enAsCas12a (dual-gRNA), CHyMErA.v2, multiSPAS were deposited to Addgene (IDs: 189632, 189633, 189634, 189635, 189636, 189637, respectively). 3Cs oligonucleotide design The protocol…

Continue Reading Optimized metrics for orthogonal combinatorial CRISPR screens

Peptide-encoding mRNA barcodes for the high-throughput in vivo screening of libraries of lipid nanoparticles for mRNA delivery

We reasoned that an ideal screening system for functional mRNA delivery would be model independent so that it could be applied in any preclinical model of disease, would consist of multiple measures of protein production that are each orthogonal to any others such that multiple formulations could be tested within…

Continue Reading Peptide-encoding mRNA barcodes for the high-throughput in vivo screening of libraries of lipid nanoparticles for mRNA delivery

Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus

CLIP enables high-efficiency knock-in into an essential locus To create CLIP, we leverage integrase-deficient lentiviruses (IDLV) to deliver an HDR donor template as a viral RNA genome to cells25,26. IDLV consists of lentiviral components but with a D64V mutation in the integrase, maintaining this enzyme’s ability to package and nuclear…

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Trends and prospects in mitochondrial genome editing

Mitochondria, double membrane-bound organelles referred to as the “powerhouses of the cell”, play an indispensable role in eukaryotic cells, as they are associated with metabolism1,2 and orchestrate a variety of other cellular functions, such as apoptosis, cell pluripotency, autophagy, calcium homeostasis, and innate immunity3,4,5,6,7,8,9. The mitochondrial genome is independent and…

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DNA double-strand break-free CRISPR interference delays Huntington’s disease progression in mice

Plasmids LentiCRISPRv2 (Addgene; 52961) was purchased from Addgene (Cambridge, MA, USA). For HTT gene suppression, LentiCRISPR v2 plasmid with U6 promoter driving sgRNA expression and EF-1 alpha promoter driving the expression of Puromycin-T2A-HA-NLS-dCas9-NLS (or Puromycin-T2A-Flag-NLS-Cas9-NLS) was used. The sgRNA-Cas9 and sgRNA-dCas9 constructs were generated by cloning the CAG repeat-targeting sgRNA…

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MISSION pLKO.1-puro Non-Target | SHC016V-1EA | SIGMA ALDRICH

Analysis Note To see more application data, protocols, vector maps visit sigma.com/shrna. Application MISSION® pLKO.1-puro Non-Target shRNA Control Transduction Particles have been used to generate 3T3-L1 (pre-adipocytes) control cell lines. General description The MISSION® pLKO.1-puro Non-Target shRNA Control Transduction Particles contain an shRNA insert that does not target any known…

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Low-grade glioma risk SNP rs11706832 is associated with type I interferon response pathway genes in cell lines

Generation and characterization of rs11706832-variant HEK293T clones A modified version of HEK293T cells where one allele of the entire LRIG1 gene has been deleted15, was used to study the potential effects of the risk SNP rs11706832 on transcriptomics and metabolomics. Sanger sequencing revealed that the single copy of rs11706832 in…

Continue Reading Low-grade glioma risk SNP rs11706832 is associated with type I interferon response pathway genes in cell lines

Efficient evolution of human antibodies from general protein language models

Acquiring amino acid substitutions via language model consensus We select amino acid substitutions recommended by a consensus of language models. We take as input a single wild-type sequence x = (x1,…,xN)∈ \(\mathcal{X}\)N, where \(\mathcal{X}\) is the set of amino acids, and N is the sequence length. We also require a set of…

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TUT4/7-mediated uridylation of a coronavirus subgenomic RNAs delays viral replication

Cell lines and tissue culture The murine 17-CL1 cell line (derived from 3T3 cells, BEI Resources, Cat. No. NR-53719) was maintained in Dulbecco Modified Eagle Medium (DMEM, Life Technologies) supplemented with 10% Fetal Bovine Serum (FBS, Gemini Bio-Products), L-glutamine (Invitrogen), and Sodium pyruvate (Invitrogen). The NCTC clone 1469 cell line…

Continue Reading TUT4/7-mediated uridylation of a coronavirus subgenomic RNAs delays viral replication

Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice

Optimizing prime editing systems for HSPCs We previously reported the use of prime editing to correct HBBS by plasmid transfection in HEK293T cells containing the SCD mutation, reaching up to 58% efficiency (Fig. 1a)24. In contrast to HEK293T cells, HSPCs are difficult to transfect with plasmid DNA but are amenable…

Continue Reading Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice

A lentiviral vector for the production of T cells with an inducible transgene and a constitutively expressed tumour-targeting receptor

Antisense vector design to accommodate independent promoters Here we sought to optimize lentivirus vector-mediated independent co-expression of two genes in transduced human T cells, with one gene under a constitutive promoter and the other under an inducible promoter, to improve adoptive T-cell transfer (ACT) of cancer. We began by building…

Continue Reading A lentiviral vector for the production of T cells with an inducible transgene and a constitutively expressed tumour-targeting receptor

A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates

IgG plasmids Antibody sequences and Fc-tagged ACE2 were cloned into the CMV/R plasmid backbone for expression under a CMV promoter. The antibodies with variable HC/LC were cloned between the CMV promoter and the bGH poly(A) signal sequence of the CMV/R plasmid to facilitate improved protein expression. The variable region was…

Continue Reading A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates

CRISPR-Cas9 for large-scale engineering

Sponsored Content by MaxCyte, Inc.Apr 13 2023Reviewed by Maria Osipova Despite progress in human genetics, individuals suffering from rare disorders, such as Duchenne muscular dystrophy (DMD), continue to face a restricted range of treatment options and a shortened lifespan. The CRISPR-Cas9 technology has enabled scientists to target specific genome regions…

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Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs

Cell culture We cultured mESCs in t2iL medium containing Dulbecco’s modified eagle medium (DMEM, Nacalai Tesque), 2 mM Glutamax (Nacalai Tesque), 1× non-essential amino acids (Nacalai Tesque), 1 mM sodium pyruvate (Nacalai Tesque), 100 U ml−1 penicillin, 100 μg ml−1 streptomycin (P/S) (Nacalai Tesque), 0.1 mM 2-mercaptoethanol (Sigma) and 15% fetal bovine serum (FBS) (Gibco), supplemented with…

Continue Reading Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs

OTUB1 promotes osteoblastic bone formation through stabilizing FGFR2

OTUB1 deficiency causes defects in osteogenesis To determine the physiological role of OTUB1 in bone homeostasis, Otub1 global knockout mice (referred to as Otub1−/−) were constructed, and their phenotypes were analyzed on embryonic (E) day 14.5 (E14.5), E16.5, E18.5, and postnatal (P) day P0 (Fig. 1a and Supplementary Fig. 1a,…

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Dopamine is involved in reparative dentin formation through odontoblastic differentiation of dental pulp stem cells

Isolation of dental pulp stem cells Human pulp cells were extracted from healthy premolars of 25-year-old female (3U) and 19-year-old male (5C) who visited Kyushu University hospital for orthodontic treatment, with informed consent as described previously29. We used CD146-positive pulp cells as DPSCs because CD146 is thought to be a…

Continue Reading Dopamine is involved in reparative dentin formation through odontoblastic differentiation of dental pulp stem cells

Collateral activity of the CRISPR/RfxCas13d system in human cells

Cell culture HEK293T, MEF-1, and U87 cell lines were used in this research. HEK293T was purchased from ATCC. MEF-1, and U87 cell lines were gifts from Muredach P. Reilly, Peter A. Sims, respectively. HEK293T, MEF-1, and U87 cells were cultured in DMEM with 4.5 g/L D-Glucose, supplemented with 10% fetal bovine…

Continue Reading Collateral activity of the CRISPR/RfxCas13d system in human cells

A Vaccinia-based system for directed evolution of GPCRs in mammalian cells

Poxvirus vectors for directed evolution of GPCRs in mammalian cells Monoclonal and homogenous expression of the gene of interest are critical prerequisites for selections in directed evolution that are difficult to achieve in mammalian cells with common expression strategies such as transient transfection. To reach both equal gene dosage and…

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Sub-millisecond conformational dynamics of the A2A adenosine receptor revealed by single-molecule FRET

Protein expression, purification and labeling The gene encoding the human A2AAR (UniProt C9JQD8) C-terminally truncated after residue Ala 316 (Supplementary Fig. 1a) was synthesized de novo (Eurofins). The nucleotide sequence was optimized for Leishmania tarentolae expression with the GeneOptimizer software (ThermoFisher Scientific). KpnI restriction site was introduced at the C-terminus and…

Continue Reading Sub-millisecond conformational dynamics of the A2A adenosine receptor revealed by single-molecule FRET

Efficient CRISPR-Cas13d-Based Antiviral Strategy to Combat SARS-CoV-2

Affiliations Expand Affiliations 1 Laboratory of Experimental Virology, Department of Medical Microbiology, Amsterdam UMC, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands. 2 Department of Molecular and Cell Biology, National Center of Biotechnology (CNB-CSIC), Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain. Free PMC article Item in…

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Base Editing Shows Promise vs. SMA

David R. Liu, PhD, a professor at Harvard University, the Broad Institute of MIT and Harvard, and the Howard Hughes Medical Institute A research team led by gene editing pioneer David R. Liu, PhD, reports the application of base editing technology to develop a one-time treatment for spinal muscular atrophy…

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CRISPR editing in the lung with novel lipids

CRISPR RNA is delivered to the mouse lung by inhalation using improved lipid nanoparticles. Many patients with inherited lung diseases lack effective treatment and could derive immense benefit from gene-editing therapies that correct the causative mutation. Direct delivery of such therapies to the lung by inhalation would be transformative, but…

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Intranasal trimeric sherpabody inhibits SARS-CoV-2 including recent immunoevasive Omicron subvariants

Research reported in this study complies with all relevant ethical regulations, and our animal study protocol has been approved by the Animal Experimental Board of Finland (license number ESAVI/28687/2020. Phage panning Sherpabody phage display library (size ~1011 cfu) was obtained from Next Biomed Therapies Oy. To develop sherpabodies specific for SARS-CoV-2…

Continue Reading Intranasal trimeric sherpabody inhibits SARS-CoV-2 including recent immunoevasive Omicron subvariants

RHOJ controls EMT-associated resistance to chemotherapy

Mice Rosa26-YFP48, Rosa-tDTomato49, K14creER, Lgr5creER (ref. 50), KrasLSLG12D (ref. 51) and Trp53fl/fl (ref. 52) mice were obtained from the NCI mouse repository and Jackson Laboratories. Rhojfl/fl mice53 were a gift from A. Uemura. All mice used in this study were composed of males and females with mixed genetic background. Mouse…

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RBFOX2 modulates a metastatic signature of alternative splicing in pancreatic cancer

Patient samples and RNA-seq analysis RNA-seq data from 395 patients with PDA was obtained from the University Health Network (Toronto), Sunnybrook Health Sciences Centre (Toronto), Kingston General Hospital (Kingston), McGill University (Montreal), Mayo Clinic (Rochester), Massachusetts General Hospital (Boston) and Sheba Medical Center (Tel Aviv) and has been described previously1,12,13,14….

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Polycystic kidney disease 2-like 1 channel contributes to the bitter aftertaste perception of quinine

Chemicals Quinine hydrochloride and denatonium benzoate were obtained from Fujifilm Wako Pure Chemical (Osaka, Japan) and dissolved in a buffer or normal water. Collagenase A and dispase II were from Roche (Basel, Switzerland). Elastase was from Worthington Biochemical (Lakewood, NJ, USA). DNase I was from Merck (Darmstadt, Germany). Lipofectamine 2000…

Continue Reading Polycystic kidney disease 2-like 1 channel contributes to the bitter aftertaste perception of quinine

Fumarate induces vesicular release of mtDNA to drive innate immunity

Mice Mice were of mixed genetic background C57BL/6 and 129/SvJ. Mice were bred and maintained under specific-pathogen-free conditions at the Breeding Unit (BRU) at the CRUK Cambridge Institute. Fh1fl/fl (ref. 4) and R26-Cre-ERT2 (ref. 5) mice were gifts from E. Gottlieb and D. Winton, respectively. Experimental mice were homozygous for…

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Viruses | Free Full-Text | Efficient CRISPR-Cas13d-Based Antiviral Strategy to Combat SARS-CoV-2

Figure 1. Replication cycle of SARS-CoV-2 and genome organization. (A) The SARS-CoV-2 replication cycle. Binding of the viral S protein to the ACE-2 receptor triggers SARS-CoV-2 infection and the plus genomic RNA (+gRNA) is translated into polyprotein (pp)1a and pp1ab. These proteins are proteolytically cleaved to generate 16 non-structural proteins…

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ANKLE1 cleaves mitochondrial DNA and contributes to cancer risk by promoting apoptosis resistance and metabolic dysregulation

ANKLE1 is the causal gene for breast and ovarian cancer risk in the chr19p13.1 region Expression quantitative trait loci (eQTL) data have revolutionized how geneticists identify candidate causal genes from genome-wide association study (GWAS) loci. We integrated the most recent meta-analysis of breast cancer GWAS10 and Genotype-Tissue Expression (GTEx) project…

Continue Reading ANKLE1 cleaves mitochondrial DNA and contributes to cancer risk by promoting apoptosis resistance and metabolic dysregulation

Nanoscale, antigen encounter-dependent, IL-12 delivery by CAR T cells plus PD-L1 blockade for cancer treatment | Journal of Translational Medicine

Lentiviral vector construction and production The lentiviral vector was designed as previously described [13, 41]. LdCV vector contains the following: human codon-optimized S. pyogenes dCas9 which was fused at the C-terminus with VP64-p65-Rta (VPR). LAT-TCS-dCas9-VPR was assembled by fusing LAT (Human cDNA, NM_001014987.2) with dCas9-VPR-Q8 and cloned into a modified…

Continue Reading Nanoscale, antigen encounter-dependent, IL-12 delivery by CAR T cells plus PD-L1 blockade for cancer treatment | Journal of Translational Medicine

RAGA prevents tumor immune evasion of LUAD by promoting CD47 lysosome degradation

RAGA regulates lung adenocarcinoma progression and macrophage phagocytosis We performed experiments to explore the role of RAGA in lung adenocarcinoma by in vitro cell culture and in vivo xenograft system. To this end, we generated RAGA knockdown A549 and H1299 cancer cells. In vitro cell proliferation assays showed that RAGA…

Continue Reading RAGA prevents tumor immune evasion of LUAD by promoting CD47 lysosome degradation

piggyBac-Mediated Genomic Integration of Linear dsDNA-Based Library for Deep Mutational Scanning in Mammalian Cells

Abstract Deep mutational scanning (DMS) makes it possible to perform massively parallel quantification of the relationship between genetic variants and phenotypes of interest. However, the difficulties in introducing large variant libraries into mammalian cells greatly hinder DMS under physiological states. Here we developed two novel strategies for DMS library construction…

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Whole-genome CRISPR activation for the identification of host factors controlling cellular interactions with SARS-CoV-2

In a recent study published in the PLOS Biology, researchers found that leucine-rich repeat-containing protein 15 (LRRC15), a toll-like receptor (TLR)-related cell surface receptor, blocked severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) binding. Study: Fibroblast-expressed LRRC15 is a receptor for SARS-CoV-2 spike and controls antiviral and antifibrotic transcriptional…

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Acquisition of new function through gene duplication in the metallocarboxypeptidase family

Recent gene duplication in the MCP gene family The MCP family of enzymes presents evidence of common ancestry, both in terms of sequence and structural homology and the chromosomal arrangement of genes. We were interested to know if any of these MCP genes continued to be duplicated more recently, and…

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SMARTchoice Lentiviral shRNA – ABO

SMARTvector Lentiviral shRNA offers maximum flexibility for vector-based RNAi experiments Because efficient gene silencing depends on both the design and level of expression of the shRNA, it is critical to choose silencing reagents where both the targeting sequence and the specific promoter driving expression are taken into consideration. SMARTvector Lentiviral…

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Whole-genome CRISPRa screening identified LRRC15 as a novel SARS-CoV-2 spike-binding protein.

(A) Schematic of CRISPRa screen used to identify regulators of SARS-CoV-2 spike binding. (B) Ranking of all genes in screen 1 by log2 fold change (LFC) calculated using MAGeCK and plotted using MAGeCKFlute. See also S1 Table. (C) Gene enrichment analysis of screen 1 performed using MAGeCK. Horizontal dotted line…

Continue Reading Whole-genome CRISPRa screening identified LRRC15 as a novel SARS-CoV-2 spike-binding protein.

LI’s Regeneron winners reflect humanity’s quest for knowledge

As founts of awe and inspiration, Long Island’s young scientists never disappoint. This month brought announcements of the 300 national semifinalists of the Regeneron Science Talent Search, the nation’s most prestigious such competition, and two weeks later, the 40 finalists who will travel to Washington in March for the last stage of the competition….

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SARS-CoV-2 WA1/2020, Delta and Omicron strains found to infect human sensory neurons

In a recent study posted to the bioRxiv* preprint server, researchers evaluated the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the peripheral nervous system (PNS). Study: Human iPS cell-derived sensory neurons can be infected by SARS-CoV-2 strain WA1/2020 as well as variants delta and omicron. Image Credit:…

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SARS-CoV-2 hijacks cellular kinase CDK2 to promote viral RNA synthesis

CDKs support SARS-CoV-2 RdRp-mediated gene expression To identify cellular proteins that modulate the SARS-CoV-2 RNA synthesis, we first performed immunoprecipitation coupled with mass spectrometry (MS) to determine cellular proteins that associate with SARS-CoV-2 RdRp. Briefly, Flag-tagged nsp7, Flag-tagged nsp8, and Flag-tagged nsp12 plasmids were co-transfected into HEK293T cells, and the…

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Unexpected finding in mucus clearance system may inform future pulmonary disease treatment

Generation and validation of MUC5B/AC-KO cultures. (A) Overview of the MUC5B/MUC5AC gene-targeting approach via lentivirus-mediated delivery of sgRNA and CRISPR-Cas9. Transduced and sorted cells were differentiated at air-liquid interface (ALI). Once fully differentiated, the mucus gel was collected by apically washing cultures. HEK293T, human embryonic kidney–293T; FACS, fluorescence-activated cell sorting;…

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Biochemical characterization of the two novel mgCas12a proteins from the human gut metagenome

Discovery of 11 new Cas12a candidates from a human gut microbiome database To discover new members of the Cas12a protein family, we built a pipeline that searches and sorts out new candidates from the National Center for Biotechnological Information (NCBI) human metagenome database. Using this search pipeline (Fig. 1a), we initially…

Continue Reading Biochemical characterization of the two novel mgCas12a proteins from the human gut metagenome