Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

Figure 1. Design of MLV-Based Gag.MS2.CRISPR/Cas9 All-in-One Chimera (A) Schematic illustration of Gag.MS2.CRISPR/Cas9 all-in-one particles. Chimeric Gag.MS2 proteins bind to non-viral RNAs containing two copies of a high-affinity MS2 target site (TS) hairpin structure. Hypothetically, redirection of the MLV-based packaging machinery should now allow specific packaging of different RNA species. In the case of CRISPR/Cas9, TS-containing Cas9 mRNA and Pol III-driven sgRNA transcripts should be co-packaged into one Gag.MS2 particle. (B) Design of the Gag.MS2 expression construct. The MLV-based wild-type Gag-Pol expression construct is depicted on top. In this plasmid, expression of Gag and Gag-Pol precursor proteins is initiated by enhancer and promoter sequences of the cytomegalovirus (CMV) and terminated by the bovine growth hormone poly(A) signal (pA). During particle maturation, Gag and Gag-Pol proteins are further processed into matrix (MA), p12, capsid (CA), NC, protease (PR), reverse transcriptase (RT), and integrase (IN) subunits. To generate the Gag.MS2 expression plasmids, coding sequences for Gag and Pol were separated on two CMV-driven plasmids. Subsequently, NC was replaced by a…

Figure 2. Individual Transfer of CRISPR/Cas9 RNAs by Gag.MS2 Particles (A) Highly efficient delivery of SpCas9.TS mRNA into target cells. Gag.MS2.SpCas9.TS particles were used to transduce NIH3T3-based RFP657.Tet2+sgRNA.Tet2 reporter cells (see the scheme above the graph) with the depicted supernatant volumes. The percentage of RFP657-negative cells was determined 6 days post-transduction. The results of 8 independent supernatants are shown. (B) Inefficient transfer of sgRNAs Tet2.TS.inc and Tet2.TS.adj by Gag.MS2 particles. RFP657.Tet2+SpCas9 NIH3T3 reporter cells were transduced with Tet2.TS.inc or Tet2.TS.adj Gag.MS2 particles. RFP657 knockout was determined by flow cytometry 6 days post-transduction. The results of 10 (Tet2.TS.inc) to 13 (Tet2.TS.adj) independently packaged supernatants are shown. (C) Co-transduction of separately packaged SpCas9.TS mRNA and sgRNA.TS resulted in poor RFP657 knockout rates. NIH3T3-based RFP657.Tet2 reporter cells were transduced with 6–8 individually packaged SpCas9.TS and Tet2.TS.inc or Tet2.TS.adj Gag.MS2 supernatants from (A) and (B) at the depicted volume ratios. Flow cytometry was performed 6 days post-transduction.

Figure 3. Efficient RFP657 Knockout by Gag.MS2.CRISPR/Cas9 All-in-One Particles SpCas9, Tet2 sgRNA, Gag.MS2, Pol, and VSVg expression plasmids were co-transfected into the HEK293T packaging cell line. The resulting supernatants SpCas9+Tet2, SpCas9.TS+Tet2.TS.inc, or SpCas9.TS+Tet2.TS.adj were harvested and concentrated 50-fold. Transfection efficiencies were determined for all produced supernatants (see also Figures S4A and S4B). (A and B) Efficient delivery of CRISPR/Cas9 RNA components depends on the presence of the TS hairpin dimer. Supernatants from HEK293T cells that exhibited transfection efficiencies <50% were used to transduce NIH3T3-based (A) or HT1080-based (B) RFP657.Tet2 reporter cells. The graphs show RFP657 knockout rates mediated by 100 μL supernatant for the individual experiments 5–6 days post-transduction. Results from 4–7 independently generated supernatants are displayed (SpCas9+Tet2 = 6 supernatants, SpCas9.TS+Tet2.TS.inc = 7 supernatants, and SpCas9.TS+Tet2.TS.adj = 4 supernatants) (see also Figure S4A). (C and D) Non-specific packaging of CRISPR/Cas9 RNAs and/or RNPs into Gag.MS2 particles. Experiments were similar to those shown in (A) and (B). However, NIH3T3 (C)…

Figure 4. Characterization of Gag.MS2.CRISPR/Cas9 Particles (A) SpCas9 and SpCas9.TS mRNA content of individually packaged Gag.MS2 particles. Absolute mRNA copies per 50 μL supernatant are shown. (B) Tet2 sgRNA quantification. The differences of incorporated Tet2 (no TS), Tet2.TS.inc, or Tet2.TS.adj sgRNAs are shown. The graph depicts the sgRNA copies per 50 μL. Absolute amounts were calculated with the help of individual standards. (C) Gag.MS2.CRISPR/Cas9 supernatants contain SpCas9 protein. SpCas9, SpCas9.TS, SpCas9.TS+Tet2.TS.inc (TS.inc), and SpCas9.TS+Tet2.TS.adj (TS.adj) Gag.MS2 supernatants were subjected to immunoblot analysis, and membranes were successively stained with Ponceau S for total, SpCas9 (163 kDa), and MS2 dimer (27 kDa) proteins. The 82 kDa band on the MS2 blot represents the immature Gag.MS2 precursor polyprotein. (D) Quantification of SpCas9 protein by ELISA within different particle types. Each data point represents one independent supernatant. (E) Chimeric Gag.MS2.CRISPR/Cas9 all-in-one particles do not integrate. The genomic DNA of transduced cells was harvested 10 days post-transduction and analyzed for the occurrence of stable integration events. The graph…

Figure 5. Stable and High-Dose SpCas9 Expression Is Cytotoxic (A) Reduced cell numbers in RIT.SpCas9-treated cultures. NIH3T3 cells were transduced with integrating RIT.EGFP or RIT.SpCas9 particles or non-integrating Gag.MS2.SpCas9.TS particles. Based on titration via real-time qRT-PCR, RIT.EGFP and RIT.SpCas9 supernatants were adjusted before transduction (see also Figure S6D). Gag.MS2.SpCas9.TS supernatants were not adjusted. Cell counts were determined 4 days post-transduction. Non-treated NIH3T3 cells served as Mock control. (B) Prolonged and high-dose SpCas9 expression decreases the metabolic activity of NIH3T3 cells. To assess the metabolic activity, transduced cells of (A) were subjected to the colorimetric MTS cell proliferation and cytotoxicity assay. The graph shows the absorbance (490 nm) of purple formazan, which originated from the reduction of MTS by cellular NAD(P)H. Results are shown as mean ± SD. (C) High-dose SpCas9 expression induces apoptosis. Transduced cells of (A) were co-stained with Annexin V and propidium iodide (PI) and analyzed for the occurrence of apoptotic (Annexin V+) and dead (PI+ or Annexin V+/PI+) cells. The left graph depicts the total percentage of…

Figure 6. Functional Knockout of Endogenous CXCR4 in Human Jurkat Cells (A) Scheme of the functional CXCR4 knockout experiment. Human Jurkat cells sorted for high CD4 and CXCR4 expression were transduced with LIT or Gag.MS2 CRISPR/Cas9 all-in-one particles for targeted CXCR4 knockout. In a second round of transduction, CXCR4 knockout cultures were transduced with a CXCR4-tropic LIT.EGFP vector. Only cells that still carry the CXCR4 receptor will be susceptible to transduction with CXCR4-tropic LIT.EGFP. The flow cytometric plots show CD4 and CXCR4 expression in Jurkat cells before and after sorting. (B) Schemes of LIT.CRISPR/Cas9.CXCR4 all-in-one and LIT.EGFP vectors. Depicted is the vector configuration after integration. All vectors are self-inactivating (SIN) due to deletion of viral enhancer and promoter sequences within the long terminal repeats (LTRs), and transgene expression (SpCas9.P2A.dTomato or EGFP) is driven by an internal spleen focus-forming virus (SFFV) promoter. In the LIT.CRISPR/Cas9.CXCR4 all-in-one vector, the hU6 promoter drives expression of sgRNA.CXCR4. Ψ, packaging signal; RRE, rev responsive element; cPPT, central polypurine tract; PRE, post-transcriptional…

Figure 7. Growth Advantage of NUFF TP53 Knockout Cells (A) Generation of DsRedexp+ NUFF cells. Freshly thawed NUFF cells (passage 9) were stably transduced with LIT.DsRedexp.IRES.Zeo encoding for DsRedexp and the Zeocin resistance gene (Zeo) at MOI 0.5. Co-expression of DsRedexp and Zeo was achieved by the internal ribosomal entry site (IRES) of endomycarditis virus and was initiated by enhancer and promoter sequences of SFFV. Transduced NUFF cells were selected with 100 μg/mL Zeocin for 10 days. The flow cytometric plots depict the percentage of DsRedexp+ NUFF cells before and after Zeocin selection. (B) Knockout of TP53, but not PTEN, resulted in a growth advantage. DsRedexp+ NUFF cells were transduced in a single round with LIT.CRISPR/Cas9 all-in-one vector particles for targeted knockout of PTEN, TP53, or Luc at MOI 10. Cells were mixed with non-treated wild-type NUFF cells 3 days post-transduction, and co-cultures were analyzed for DsRedexp expression for up to 67 days. The control co-culture of non-treated DsRedexp+ and wild-type NUFF cells is marked in red. Exp 1, experiment 1; Exp 2, experiment 2. (C) Outgrowth of DsRedexp+ NUFF cells that were treated with Gag.MS2.CRISPR/Cas9.TP53…