Guide-it recombinant Cas9

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Knock out of CD81 in induced pluripotent stem cells

Knock out of CD81 in induced pluripotent stem cells. In this experiment, two induced pluripotent stem cell lines, hiPSC-18 and hiPSC-22, were electroporated withCas9/sgRNA RNPs against CD81. Cells were stained for CD81 and then run on a FACS machine to detect CD81 ten days after editing. The negative staining control shows the FACS data from cells without antibody staining, while the electroporation control shows cells electroporated without Cas9/sgRNA RNPs. When cells were electroporated with both Cas9 and sgRNA, very high editing efficiency was observed.

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Homology-directed repair at the AAVS1 or CXCR4 genes

Homology-directed repair at the AAVS1 or CXCR4 genes. Panel A. These diagrams demonstrate how the HDR experiments in CD34+ HSCs were done. A ssdNA template containing a HindIII restriction site was inserted into the AAVS1 gene, and a template containing both HindIII and BamHI restriction sites was inserted into the CXCR4 gene. The homology arms were 90 bp on each side. Panel B. After electroporation of the repair template and Cas9/sgRNA RNPs, gene targets were amplified from target cells using PCR and analyzed by restriction digest. Editing efficiencies are shown above each positive well on the gel. The extra band in the BamHI digest of the CXCR4 gene is due to a second BamHI site already present in the wild-type gene before editing. We verified that 98% of the HSCs stained positive for CD34+ five days after editing.

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Gene knockouts in the Cellartis hiPSC-18 cell line

Gene knockouts in the Cellartis hiPSC-18 cell line. The strength of the cleaved bands gives a semiquantitative estimate of the percentage of edited cells. As can be seen, the Guide-it recombinant Cas9, when combined with sgRNA from the Guide-it In Vitro Transcription Kit, provides consistent and effective gene editing for many targets when compared with other vendors’ recommended methods for producing guide RNAs. Numbers at the bottom of each gel represent gene editing efficiencies (expressed as a %) for the indicated RNPs.

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Knockout of therapeutically relevant genes in CD3+ Pan T cells

Knockout of therapeutically relevant genes in CD3+ Pan T cells. In this experiment, CD3+ Pan T cells were electroporated with Cas9/sgRNA RNPs in order to perform single (B2M or PD-1) or double knockouts (B2M+PD-1 or TRAC+TRBC). After thawing, cells were grown in LymphoONE medium and activated with ImmunoCult Human CD3/CD28/CD2 T Cell Activator for three days. Cells were then electroporated with RNP complexes in triplicate, following the Guide-it Recombinant Cas9 (10 μg/μl) User Manual. For the double knockouts, RNP complexes targeting the respective genes were prepared independently and mixed with the cells just before electroporation. Seven days after electroporation, knockout frequencies were measured by flow cytometry.

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Knockout of B2M in CD3+ Pan T cells

Knockout of B2M in CD3+ Pan T cells. In this experiment, CD3+ Pan T cells were electroporated with Cas9/sgRNA RNPs in order to knockout B2M. After thawing, cells were grown in LymphoONE medium and activated with ImmunoCult Human CD3/CD28/CD2 T Cell Activator for three days. Cells were then electroporated with RNP complexes in triplicate, according to the Guide-it Recombinant Cas9 (10 μg/μl) User Manual. Seven days after electroporation, knockout frequencies and cell viability were measured by flow cytometry.

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632679: Guide-it Recombinant Cas9 (10 µg/µl)

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632678: Guide-it Recombinant Cas9 (10 µg/µl)

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Knock out of CD81 in induced pluripotent stem cells

Knock out of CD81 in induced pluripotent stem cells. In this experiment, two induced pluripotent stem cell lines, hiPSC-18 and hiPSC-22, were electroporated withCas9/sgRNA RNPs against CD81. Cells were stained for CD81 and then run on a FACS machine to detect CD81 ten days after editing. The negative staining control shows the FACS data from cells without antibody staining, while the electroporation control shows cells electroporated without Cas9/sgRNA RNPs. When cells were electroporated with both Cas9 and sgRNA, very high editing efficiency was observed.

.

Back

Homology-directed repair at the AAVS1 or CXCR4 genes

Homology-directed repair at the AAVS1 or CXCR4 genes. Panel A. These diagrams demonstrate how the HDR experiments in CD34+ HSCs were done. A ssdNA template containing a HindIII restriction site was inserted into the AAVS1 gene, and a template containing both HindIII and BamHI restriction sites was inserted into the CXCR4 gene. The homology arms were 90 bp on each side. Panel B. After electroporation of the repair template and Cas9/sgRNA RNPs, gene targets were amplified from target cells using PCR and analyzed by restriction digest. Editing efficiencies are shown above each positive well on the gel. The extra band in the BamHI digest of the CXCR4 gene is due to a second BamHI site already present in the wild-type gene before editing. We verified that 98% of the HSCs stained positive for CD34+ five days after editing.

.

Back

Gene knockouts in the Cellartis hiPSC-18 cell line

Gene knockouts in the Cellartis hiPSC-18 cell line. The strength of the cleaved bands gives a semiquantitative estimate of the percentage of edited cells. As can be seen, the Guide-it recombinant Cas9, when combined with sgRNA from the Guide-it In Vitro Transcription Kit, provides consistent and effective gene editing for many targets when compared with other vendors’ recommended methods for producing guide RNAs. Numbers at the bottom of each gel represent gene editing efficiencies (expressed as a %) for the indicated RNPs.

Back

Knockout of therapeutically relevant genes in CD3+ Pan T cells

Knockout of therapeutically relevant genes in CD3+ Pan T cells. In this experiment, CD3+ Pan T cells were electroporated with Cas9/sgRNA RNPs in order to perform single (B2M or PD-1) or double knockouts (B2M+PD-1 or TRAC+TRBC). After thawing, cells were grown in LymphoONE medium and activated with ImmunoCult Human CD3/CD28/CD2 T Cell Activator for three days. Cells were then electroporated with RNP complexes in triplicate, following the Guide-it Recombinant Cas9 (10 μg/μl) User Manual. For the double knockouts, RNP complexes targeting the respective genes were prepared independently and mixed with the cells just before electroporation. Seven days after electroporation, knockout frequencies were measured by flow cytometry.

Back

Knockout of B2M in CD3+ Pan T cells

Knockout of B2M in CD3+ Pan T cells. In this experiment, CD3+ Pan T cells were electroporated with Cas9/sgRNA RNPs in order to knockout B2M. After thawing, cells were grown in LymphoONE medium and activated with ImmunoCult Human CD3/CD28/CD2 T Cell Activator for three days. Cells were then electroporated with RNP complexes in triplicate, according to the Guide-it Recombinant Cas9 (10 μg/μl) User Manual. Seven days after electroporation, knockout frequencies and cell viability were measured by flow cytometry.