The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo. So far, the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here, we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. We developed 6 strains of genome-modified mice using CRISPR/Cas9 system, and utilized this approach to genotype mice from F0 to F2 generation, which included single and multiplexed genome-modified mice. We also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%. We further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus, PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN.

T20 (generic name: Enfuvirtide, brand name: Fuzeon) is the only FDA-approved HIV fusion inhibitor that is being used for treatment of HIV/AIDS patients who have failed to respond to current antiretroviral drugs. However, it rapidly induces drug resistance in vitro and in vivo. On the basis of the structural and functional information of anti-HIV peptides from a previous study, we designed an HIV fusion inhibitor named CP32M, a 32-mer synthetic peptide that is highly effective in inhibiting infection by a wide range of primary HIV-1 isolates from multiple genotypes with R5-or dual-tropic (R5X4) phenotype, including a group O virus (BCF02) that is resistant to T20 and C34 (another anti-HIV peptide). Strikingly, CP32M is exceptionally potent (at low picomolar level) against infection by a panel of HIV-1 mutants highly resistant to T20 and C34. These findings suggest that CP32M can be further developed as an antiviral therapeutic against multidrug resistant HIV-1.drug-resistance ͉ gp41 ͉ peptide ͉ six-helix bundle

Human immunodeficiency virus type 1 (HIV-1) entry into the host cell involves a cascade of events and currently represents one of most attractive targets in the search for new antiviral drugs. The fusion-active gp41 core structure is a stable six-helix bundle (6-HB) folded by its trimeric N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR). Peptides derived from the CHR region of HIV-1 gp41 are potent fusion inhibitors that target the NHR to block viral and cellular membrane fusion in a dominant negative fashion. However, all CHR peptides reported to date are derived primarily from residues 628 to 673 of gp41; little attention has been paid to the upstream sequence of the pocket binding domain (PBD) in the CHR. Here, we have identified a motif ( 621 QIWNNMT 627 ) located at the upstream region of the gp41 CHR, immediately adjacent to the PBD ( 628 WMEWEREI 635 ). Biophysical characterization demonstrated that this motif is critical for the stabilization of the gp41 6-HB core. The peptide CP621-652, containing the 621 QIWNNMT 627 motif, was able to interact with T21, a counterpart peptide derived from the NHR, to form a typical 6-HB structure with a high thermostability (thermal unfolding transition [T m ] value of 82°C). In contrast, the 6-HB formed by the peptides N36 and C34, which has been considered to be a core structure of the fusion-active gp41, had a T m of 64°C. Different from T-20 (brand name Fuseon), which is the first and only HIV-1 fusion inhibitor approved for clinical use, CP621-652 could efficiently block 6-HB formation in a dose-dependent manner. Significantly, CP621-652 had potent inhibitory activity against HIV-1-mediated cell-cell fusion and infection, especially against T-20-and C34-resistant virus. Therefore, our works provide important information for understanding the core structure of the fusion-active gp41 and for designing novel anti-HIV peptides.The entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by the attachment of its envelope (Env) glycoprotein to cell surface receptors. The Env glycoprotein, a type I transmembrane protein, is originally synthesized as a single, glycosylated, polyprotein precursor, gp160, which is believed to assemble a trimeric structure in the endoplasmic reticulum and is subsequently cleaved by a cellular protease to yield a surface subunit, gp120, and a transmembrane subunit, gp41 (23, 53). gp120 is responsible for virus binding to its cell receptor, CD4, and a coreceptor (CRR5 or CXCR4). gp41 mediates membrane fusion of the virus with the target cell (45). Like other type I transmembrane proteins, the gp41 molecule consists of extracellular, transmembrane, and cytoplasmic domains (Fig. 1A). Its extracellular domain (ectodomain) contains four major functional regions: a hydrophobic, glycine-rich fusion peptide (FP), an N-terminal heptad repeat (NHR) (or HR1), a C-terminal heptad repeat (CHR) (or HR2), and a tryptophan-rich region. Both the NHR and CHR contain 4-3 repeats of hydrophobic amino acids predicted to f...

No abstract

No abstract

Structures could be achieved when proteins are overlapped with surroundings free of tilt series -The particle detection efficiency is significantly improved -Allowing the usage of homolog proteins as templates -The throughput of structure determination is remarkably enhanced ll www.cell.com/the-innovation

The outbreak of COVID-19 has so far inflicted millions of people all around the world and will have a long lasting effect on every aspect of everyone's life. Yet there is no effective approved treatment for the disease. In an effort of utilizing human ferritin as nanoplatform for drug delivery, we engineered a fusion protein by presenting receptor-binding motif (RBM) of SARS-CoV-2 virus spike glycoprotein on the N-terminus of ferritin subunits. The designed fusion protein with a cage-like structure, similar to that of corona virus, is a potential anti-SARS-CoV-2 vaccine. We hereby show the construction, preparation, and characterization of the fusion protein RBM-HFtn. Our initial affinity study confirmed its biological activity towards ACE2 receptor which suggests its mode of action against SARS-CoV-2 could be either through vaccine therapy or blocking the cellular entry of virus as antagonist of ACE2 receptor.

No abstract