Journal: The Journal of Biological Chemistry

Article Title: Recharacterization of the mammalian cytosolic type 2 ( R )-β-hydroxybutyrate dehydrogenase as 4-oxo- l -proline reductase (EC 1.1.1.104)

doi: 10.1016/j.jbc.2022.101708

Figure Lengend Snippet: Western blot analysis of BDH2 expression in four Bdh2 -deficient HEK293T clonal cell lines ( Bdh2 -KO) and WT HEK293T cells (WT). The KO clonal cell lines (A12, A15, B9, and B13) were generated by the CRISPR–Cas9 gene-inactivation procedure. The Western blot analysis was carried out using 40 μg of the cell lysate protein, a primary rabbit antibody against the human BDH2 (catalog no.: PA5-44760; Invitrogen), and a horseradish peroxidase–conjugated goat anti-rabbit secondary antibody. The secondary antibody was detected by measuring enhanced chemiluminescence. The presence of a nonspecific signal (≈30 kDa) is in agreement with the specification of the primary antibody. BDH2, type 2 ( R )-β-hydroxybutyrate dehydrogenase; HEK293T, human embryonic kidney 293T cell line.

Article Snippet: This vector encodes an enhanced specificity Cas9 enzyme (a gift from Andrea Németh; Addgene; plasmid no.: 101039) ( ).

Techniques: Western Blot, Expressing, Generated, CRISPR

Journal: Microbial Biotechnology

Article Title: Developing a CRISPR‐assisted base‐editing system for genome engineering of Pseudomonas chlororaphis

doi: 10.1111/1751-7915.14075

Figure Lengend Snippet: Design of a CRISPR‐assisted base‐editing system tailored for Pseudomonas chlororaphis . A. Schematic illustration of the plasmid‐borne base‐editing system. The tool consists of an enhanced specificity Cas9 nickase (eSpCas9pp D10A ), fused to a cytidine deaminase (rAPOBEC1, which catalyses the C‐to‐T conversion), and the uracil DNA glycosylase inhibitor (UGI). The base‐editing machinery is directed to the target DNA by a single guide RNA (sgRNA). PAM , protospacer adjacent motif. B. Biochemical reaction catalysed by the cytidine deaminase rAPOBEC1. C. Structure of plasmid pBRC1, containing the construct that encodes the SpCas9pp D10A , rAPOBEC1 and UGI fusion. This module is under transcriptional control by the L‐arabinose‐inducible AraC/ P araBAD system, whereas the sgRNA is constitutively expressed from a P lac promoter. XTEN, short peptide linker sequence with no specific structure; N 20 , specific 20‐nt sequence targeting the gene to be edited; and Km R , kanamycin resistance. D. Strategy followed for gene inactivation by introducing premature STOP codons (TAA, TAG and TGA) in target genes using the base‐editing system. UGI inhibits uracil base excision on the non‐template DNA strand, so that DNA repair occurs on the template strand.

Article Snippet: A DNA cassette encoding the cytidine deaminase from rat [rAPOBEC1; Komor et al . ( )], an enhanced specificity Cas9 nickase [eSpCas9pp D10A , carrying the point mutations K848A, K1003A, R1060A and D10A; Sun et al . ( )], a XTEN linker [a short connector sequence; Komor et al . ( )] and the uracil DNA glycosylase inhibitor (UGI) was amplified from plasmid pSEVA2BE [a derivative of standard vector pSEVA644; Sun et al . ( )] and assembled in plasmid pBRC1 (deposited in Addgene, ID 183064).

Techniques: CRISPR, Plasmid Preparation, Construct, Control, Sequencing

Journal: Nucleic Acids Research

Article Title: Highly efficient ‘hit-and-run’ genome editing with unconcentrated lentivectors carrying Vpr.Prot.Cas9 protein produced from RRE-containing transcripts

doi: 10.1093/nar/gkaa561

Figure Lengend Snippet: Delivery of the first generation of the two-component lentivector nanoparticles carrying the Cas9 nuclease protein and a template for the U6-sgRNA expression cassette (VECTR-Cas(sgGFP)) to human HEK293-EGFP cells. ( A ) Design of the constructs to generate the lentivector particles. Cas9 was fused to the C-terminus of Vpr containing an authentic HIV-1 protease cleavage site (CTLNF/PISPI; Vpr.Prot.Cas9). The U6-sgRNA expression cassette was incorporated into a lentiviral expression vector (Lenti(sgRNA)). The packaging construct (psPAX2) encodes the structural and enzymatic components of virions. The VSV.G envelope protein was used to pseudotype and stabilize viral particles (pHCMV-G). Efficient nuclear export and colocalization of mRNA for translation were supported by adding the Rev-responsive element (RRE) to the constructs and by overexpressing Rev during virion production (pRSV-Rev). Gag-Pol subunits: matrix (MA), capsid (CA), nucleocapsid (NC), p6, protease (PR), reverse transcriptase (RT) and integrase (IN). Packaging signal (ψ); promoters (CMV, CAG, RSV, U6 and EFS), polyadenylation signal (pA), posttranscriptional regulatory element (WPRE). ( B ) The EGFP gene disruption in HEK293-EGFP cells after transduction with the first generation of the two-component lentivector (VECTR-Cas(sgGFP); red entry) or a control LentiCRISPRv2(sgGFP) (blue entry).

Article Snippet: The presence of viral and heterologous proteins in the cell lysates (cell, 10 μg of total protein) and virions, harvested from cell culture supernatant 48 h after transfection (virus, 100× concentrated by ultracentrifugation [20 000 g, 4°C, 2 h]), was determined by immunoblotting with antibodies specific for Cas9 protein (α-Cas9 [mouse, 1:1000, 7A9-3A3; Cell Signalling Technology]).

Techniques: Expressing, Construct, Plasmid Preparation, Reverse Transcription, Disruption, Transduction, Control

Journal: Nucleic Acids Research

Article Title: Highly efficient ‘hit-and-run’ genome editing with unconcentrated lentivectors carrying Vpr.Prot.Cas9 protein produced from RRE-containing transcripts

doi: 10.1093/nar/gkaa561

Figure Lengend Snippet: Enhanced packaging of the Vpr.Prot.Cas9 fusion protein produced from the transcript containing the Rev responsive element (RRE). ( A ) Schematic representation of the expression cassettes encoding Vpr.Prot.Cas9 fusion protein. The transcripts encoding Cas9 contain either the Rev responsive element (RRE) or the constitutive transport element (CTE). ( B ) The constructs were co-transfected into HEK293T cells together with psPAX2 (encodes structural and enzymatic components of virions from transcript containing the RRE) and with the other plasmids required for virus production as described in the Figure . The presence of viral and heterologous proteins in the cell lysates (cell) and virions (virus) harvested from cell culture supernatant 48 h after transfection was determined by immunoblotting with antibodies as follows. The blot was probed with an antibody specific for the Cas9 protein. Next, the membrane was washed and re-probed with an antibody detecting the p24 (CA) and Pr55 (Gag) proteins to monitor the expression of the viral structural proteins in the lysates and to determine the amounts of virions released from transfected cells. Equivalent loading was confirmed by re-probing with an antibody directed against the HSP90 protein and by Coomassie blue staining of the SDS-PAGE gel after blotting. One representative example from three biological replicates performed in three different weeks is shown; 293T, untransfected cells; M, Color Prestained Standard NEB #P7712. ( C ) A model describing how nuclear export functions affect the packaging of heterologous proteins into virions. Retrovirus assembly and budding is a highly concerted process. It is mediated by numerous, largely undefined spatially and temporally regulated interactions between viral proteins and cellular factors. Previous reports showed that the regulation of the HIV-1 Gag assembly begins as soon as nuclear export factors are deposited onto the transcripts encoding the structural components of HIV-1. Here, we present a model whereby the selection of RNA export pathway modulates the cytosolic fate and function of the transcripts encoding heterologous proteins and facilitates the packaging of non-viral proteins into virions. The nuclear export of both viral and non-viral transcripts via the same pathway facilitates the cytoplasmic co-localization of the transcripts and their translation products. The close proximity of Gag and Vpr.Prot.Cas9 promotes the interaction between the two polyproteins that is required for encapsidation of the fusion protein into virions.

Article Snippet: The presence of viral and heterologous proteins in the cell lysates (cell, 10 μg of total protein) and virions, harvested from cell culture supernatant 48 h after transfection (virus, 100× concentrated by ultracentrifugation [20 000 g, 4°C, 2 h]), was determined by immunoblotting with antibodies specific for Cas9 protein (α-Cas9 [mouse, 1:1000, 7A9-3A3; Cell Signalling Technology]).

Techniques: Produced, Expressing, Construct, Transfection, Virus, Cell Culture, Western Blot, Membrane, Staining, SDS Page, Selection

Journal: Nucleic Acids Research

Article Title: Highly efficient ‘hit-and-run’ genome editing with unconcentrated lentivectors carrying Vpr.Prot.Cas9 protein produced from RRE-containing transcripts

doi: 10.1093/nar/gkaa561

Figure Lengend Snippet: Delivery of the second (VECTRv2-Cas(sgGFP)) or third (VECTRv3-Cas(sgGFP)) generation of the two-component lentivector nanoparticles to human HEK293-EGFP cells. ( A ) The EGFP gene disruption in HEK293-EGFP cells after transduction with the second generation of the two-component lentivector (VECTRv2-Cas(sgGFP); red entries) or a control LentiCRISPRv2(sgGFP) (blue entry). ( B ) T7 endonuclease I (T7EI) assay to measure the indels in the EGFP gene resulting from transduction with the VECTRv2-Cas(sgGFP), the same vector lacking Vpr.Prot.Cas9 or a control vector pLentiCRISPRv2(sgGFP). The frequency of indel formation was calculated as described in the methods section. Please note that it may decrease the actual editing efficiency for highly efficient editing. ( C ) Mutant sequences at the EGFP locus and their frequencies, as determined by SYNTHEGO analysis of Sanger sequencing of a PCR product amplified from VECTRv2-Cas(sgGFP)-transduced HEK293-EGFP cells. The 20-nt target sequence is shown with a blue background. The protospacer adjacent motif (PAM) sequence is shown in blue. ( D ) Comparison of EGFP disruption after transduction with lentiviral particles containing integration-deficient (D64V; VECTRv3-Cas(sgGFP)) or integration-proficient (WT; VECTRv2-Cas(sgGFP)) integrase. ( E ) Time-course analysis of EGFP disruption mediated by the VECTRv3-Cas(sgGFP) or the gene-delivering LentiCRISPRv2(sgGFP). ( F ) A schematic representation of lentivector-mediated delivery of the Cas9 protein and viral RNA containing U6-sgRNA. Cas9 is packaged into virions as a Vpr.Prot.Cas9 fusion polyprotein that is proteolytically cleaved during virion maturation . Following virus entry into a recipient cell , the viral genome is reverse transcribed to DNA and translocated to the nucleus together with Cas9 , where the U6 promoter drives the expression of sgRNA . The nascent sgRNA associates with Cas9 and directs the nuclease to the target site in the genomic DNA (gDNA) for cleavage . (A, D) The mean activities of three replicates are shown. (E) The mean of two replicates are shown. (A, D, E) Error bars, mean ± s.e.m.

Article Snippet: The presence of viral and heterologous proteins in the cell lysates (cell, 10 μg of total protein) and virions, harvested from cell culture supernatant 48 h after transfection (virus, 100× concentrated by ultracentrifugation [20 000 g, 4°C, 2 h]), was determined by immunoblotting with antibodies specific for Cas9 protein (α-Cas9 [mouse, 1:1000, 7A9-3A3; Cell Signalling Technology]).

Techniques: Disruption, Transduction, Control, T7EI Assay, Plasmid Preparation, Mutagenesis, Sequencing, Amplification, Comparison, Virus, Reverse Transcription, Expressing

Journal: European Journal of Endocrinology

Article Title: The phenotypic spectrum associated with OTX2 mutations in humans

doi: 10.1530/EJE-20-1453

Figure Lengend Snippet: OTX2 H230L had no abnormal phenotype, and C-terminal variants are tolerated. (A) CRISPR/Cas9 generated Otx2 H230L and multiple by-product variants. (B) Otx2 H230L had no obvious phenotype in either the pituitary gland or the eye. Otx2 L219fs*17 showed anophthalmia/microphthalmia but, however, showed similar pituitary morphology to WT animals. Arrows and arrowheads indicate the pituitary gland and optic nerve, respectively. (C) HE staining of eyes of adult mice. Otx2 H230L had normal eyes, but the retina of Otx2 L219fs*17 was thin. (D, E and F) Pituitary histology of P0 mice. (D) HE staining, (E) GH staining, and (F) LH staining were similar between genotypes.

Article Snippet: The Otx2 variant alleles were generated by microinjecting enhanced specificity Cas9 protein (30 ng/uL, Integrated DNA Technologies), DNA donor oligo (10 ng/µL, Integrated DNA Technologies), and C77G2 crRNA 10 ng/μL annealed with tracrRNA 15 ng/μL (Integrated DNA Technologies) into fertilized eggs obtained from super-ovulated B6CBAF1 females purchased from the Jackson Laboratory.

Techniques: CRISPR, Generated, Staining

Journal: EMBO Reports

Article Title: Targeting monocytic Occludin impairs transendothelial migration and HIV neuroinvasion

doi: 10.1038/s44319-024-00190-x

Figure Lengend Snippet: ( A ) Schematic representation of a CRISPR/Cas9 lentiviral construct used for the generation of OCLN KO THP-1 cells with indicated target sequences. ( B ) Western Blot analysis of OCLN expression in indicated cell lines. Actin is used as a loading control. The hCMEC/D3 and THP-1 cells express OCLN (two bands), the THP-1 OCLN KO cells do not show OCLN, although non-specific bands of lower size and very weak intensity can be observed. As controls, NIH 3T3 do not express OCLN and THP-1 WT transduced with EGFP-OCLN express both endogenous and overexpressed OCLN. ( C ) Western Blot analysis of OCLN expression in THP-1 OCLN KO cells. GAPDH is used as a loading control. EGFP-OCLN can be seen with both anti-Occludin and anti-GFP antibody (green arrow at EGFP). EGFP-OCLN-ΔC and EGFP-CAAX can be seen with anti-GFP antibody only (red and yellow arrows, respectively). ( D ) Mean fluorescence intensity of EGFP-CAAX, EGFP-OCLN, or EGFP-OCLN-ΔC expressed by THP-1 OCLN KO cells measured by flow cytometry. The data were normalized to EGFP-CAAX obtained from n = 3 individual experiments. Each symbol’s color corresponds to an individual experiment. ( E ) Western Blot showing endogenous OCLN expression in human primary monocytes and exogenous expression of EGFP-OCLN in the same donors (2 donors). GAPDH is used as a loading control. ( F ) Immunofluorescence images of primary monocytes transduced with EGFP-OCLN, EGFP-CAAX, or non-transduced, fixed and stained with anti-OCLN antibody (cyan) and DAPI (yellow). EGFP is in magenta. Scale bar: 2 μm. ( G ) Efficiency of transduction of primary monocytes from 2 donors performed in two technical replicates measured by flow cytometry. ( H ) Mean fluorescence intensity of EGFP-CAAX, EGFP-OCLN or EGFP-OCLN-ΔC expressed by primary monocytes. The data were obtained from n = 6 monocyte donors normalized to EGFP-CAAX of each experiment. Each symbol’s color corresponds to an individual monocyte donor. In ( D , G , H ), the data are presented as mean ± SEM. .

Article Snippet: First, we built a lentivector expressing 2 guide RNAs (gRNAs) targeting OCLN, a Cas9-specific tracrRNA and the Cas9 enzyme (Fig. , and Addgene #208398).

Techniques: CRISPR, Construct, Western Blot, Expressing, Control, Transduction, Fluorescence, Flow Cytometry, Immunofluorescence, Staining