cas9 coding sequence (Integrated DNA Technologies)

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Integrated DNA Technologies cas9 coding sequence

Delivery of the first generation of the two-component lentivector nanoparticles carrying the <t>Cas9</t> 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).

Cas9 Coding Sequence, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 4157 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Highly efficient ‘hit-and-run’ genome editing with unconcentrated lentivectors carrying Vpr.Prot.Cas9 protein produced from RRE-containing transcripts"

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

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkaa561

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).

Figure Legend 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).

Techniques Used: Expressing, Construct, Plasmid Preparation, Transduction

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.

Figure Legend 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.

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

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.

Figure Legend 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.

Techniques Used: Transduction, T7EI Assay, Plasmid Preparation, Mutagenesis, Sequencing, Amplification, Expressing

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cas9 coding sequence (Integrated DNA Technologies)

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