ht1080 egfp reporter cells  (InvivoGen)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells

doi: 10.1016/j.omtn.2021.12.033

Figure Lengend Snippet: Gammaretrovirus-based Gag.MS2 chimera for CRISPR-Cas9 delivery (A) Design of the gammaretroviral Gag.MS2 (g.Gag.MS2) variant and non-retroviral SpCas9.TS and sgRNA.TS expression plasmids. The configuration of structural Gag and enzymatic Pol proteins within gammaretroviral wt Gag-Pol (wt g.Gag-Pol) is depicted at the top. It consists of matrix (MA), p12, capsid (CA), nucleocapsid (NC), protease (PR), reverse transcriptase (RT), and integrase (IN) subdomains, which are separated from each other by individual protease sites. In g.Gag.MS2, the MS2CP dimer (2× MS2CP) protein replaces NC while maintaining the natural retroviral protease site (black bold bar). To ensure specific packaging of CRISPR-Cas9 RNA into assembling g.Gag.MS2 particles, two copies of MS2 target site (TS) are incorporated downstream of EGFP within the SpCas9.TS expression plasmid or were placed in (TS.inc) or adjacent (TS.adj) to the sgRNA scaffold in respective sgRNA expression plasmids. Co-expression of EGFP and DsRedexp helped to monitor transfection during particle production. (B) Direct comparison of integrating (g.RIT) and non-integrating g.Gag.MS2 CRISPR.Tet2 particles. Indicated supernatant volumes were used to transduce human HT1080-based RFP657.Tet2 reporter cells. Each data point represents one individually generated supernatant (n = 3). CMV, promoter from cytomegalovirus; hU6, human RNA Pol III U6 promoter; pA, poly(A) signal; PRE, post-transcriptional regulatory element from woodchuck hepatitis virus; SFFV, promoter from spleen focus forming virus.

Article Snippet: HT1080 RFP657.Tet2 and HT1080 EGFP reporter cells were additionally cultured in the presence of 1 μg/mL puromycin (InvivoGen, Toulouse, France).

Techniques: CRISPR, Variant Assay, Retroviral, Expressing, Reverse Transcription, Plasmid Preparation, Transfection, Comparison, Transduction, Generated, Virus

Journal: Molecular Therapy. Nucleic Acids

Article Title: Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells

doi: 10.1016/j.omtn.2021.12.033

Figure Lengend Snippet: Alpharetrovirus-based Gag.MS2 particles showed improved delivery of CRISPR-Cas9 components into target cells (A) Design of alpharetroviral Gag.MS2 (a.Gag.MS2) variants. The wt alpharetroviral Gag-Pol polyprotein (wt a.Gag-Pol) is shown at the top of the figure panel. In contrast to g.Gag-Pol, alpharetroviral PR is part of the Gag open reading frame (ORF) (and not Pol ORF). In the depicted a.Gag.MS2 variant, the MS2CP dimer was separated from NC by the naturally occuring viral protease site (black bold bar). (B) a.Gag.MS2.CRISPR.Tet2 particles containing the Tet2.TS.inc sgRNA variant depict higher knockout rates compared with their Tet2.TS.adj counterparts. Indicated volumes of supernatants were used to transduce HT1080-based RFP657.Tet2 reporter cells. The graph depicts RFP657.Tet2 knockout rates mediated by three independent supernatants (n = 3). (C) a.Gag.MS2.CRISPR.Tet2 particles are superior to their gammaretroviral counterparts. Direct comparison of three individually produced a.Gag.MS2- and g.Gag.MS2-based CRISPR.Tet2 supernatants in HT1080-based RFP657.Tet2 reporter cells is shown (n = 3). (D) a.Gag.MS2.CRISPR.Tet2 particles mediate efficient targeted gene knockout in hiPSCs. hiPSC-based RFP657.Tet2 reporter cells were transduced with indicated volumes of three different batches of g.Gag.MS2.CRISPR.Tet2 or a.Gag.MS2.CRISPR.Tet2 supernatants (n = 3). A LIT.CRISPR.Tet2 supernatant applied at MOI 2 (2.9 μL) or MOI 5 (7.4 μL) served as a positive control (n = 1).

Article Snippet: HT1080 RFP657.Tet2 and HT1080 EGFP reporter cells were additionally cultured in the presence of 1 μg/mL puromycin (InvivoGen, Toulouse, France).

Techniques: CRISPR, Variant Assay, Knock-Out, Transduction, Comparison, Produced, Gene Knockout, Positive Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells

doi: 10.1016/j.omtn.2021.12.033

Figure Lengend Snippet: Characterization of alpha- and gammaretroviral Gag.MS2.CRISPR.Tet2 supernatants In total, two different batches (white and black filled circles), each with three individually packaged supernatants for a.Gag.MS2 and g.Gag.MS2 CRISPR.Tet2 particles, were generated and characterized for their content of CRISPR-Cas9 components (n = 6). (A) SpCas9.TS mRNA and sgRNA.TS content in a.Gag.MS2- and g.Gag.MS2-based CRISPR.Tet2 supernatants. Total RNA copies per microliter supernatant were calculated using individual plasmid standards. (B) Gag.MS2.CRISPR.Tet2 supernatants contain SpCas9 protein. SpCas9 protein concentrations were assessed by an SpCas9 ELISA. (C) a.Gag.MS2.CRISPR.Tet2 supernatants had a higher Gag.MS2 precursor protein content. Immunoblot analysis of three individually packaged a.Gag.MS2.CRISPR.Tet2 and g.Gag.MS2.CRISPR.Tet2 supernatants is shown (batch 2; black filled circles) (n = 3). g.Gag.MS2 (82 kDa) and a.Gag.MS2 (89 kDa) precursor proteins were detected with an anti-MS2 antibody. Ponceau S staining of the membrane is shown below. (D) g.Gag.MS2.CRISPR.Tet2-mediated RFP657.Tet2 knockout rates can be adjusted by ∼10-fold higher supernatant volumes. Characterized supernatants from batch 2 were used to transduce HT1080 RFP657.Tet2 reporter cells at the depicted volumes (n = 3). (E) Normalization of supernatant volumes showed an ∼2-fold-higher potency for a.Gag.MS2-based CRISPR.Tet2 particles. The in (D) acquired datasets were replotted as % RFP657.Tet2 knockout against applied SpCas9.TS mRNA copies/cell (n = 3; mean values are depicted).

Article Snippet: HT1080 RFP657.Tet2 and HT1080 EGFP reporter cells were additionally cultured in the presence of 1 μg/mL puromycin (InvivoGen, Toulouse, France).

Techniques: CRISPR, Generated, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Membrane, Knock-Out, Transduction

Journal: Molecular Therapy. Nucleic Acids

Article Title: Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells

doi: 10.1016/j.omtn.2021.12.033

Figure Lengend Snippet: Efficient CRISPR-mediated transition of EGFP to EBFP by a.Gag.MS2.CRISPR.HDR particles (A) Determined copies of CRISPR RNA and PT-modified ssODN molecules within a.Gag.MS2.CRISPR.HDR supernatants. (B and C) HT1080-based (B) or NuFF-based (C) EGFP reporter cells were transduced with 30 μL (1,980 copies SpCas9.TS mRNA/cell) of a.Gag.MS2.CRISPR.HDR supernatants in the absence (w/o) or presence of NHEJ or HDR inhibitors (inh) M3814 or YU238259, respectively. DMSO-treated cells served as solvent controls. The graphs display % EBFP + cells within treated cultures. Non-treated or DMSO-treated mock cultures indicate the degree of autofluorescence in the EBFP channel. Each data point reflects one independently prepared supernatant (n = 3–4).

Article Snippet: HT1080 RFP657.Tet2 and HT1080 EGFP reporter cells were additionally cultured in the presence of 1 μg/mL puromycin (InvivoGen, Toulouse, France).

Techniques: CRISPR, Modification, Transduction, Solvent