Takara Guide-it Recombinant Cas9 (10 µg/µl) \200 ug

Guide-it Recombinant Cas9 (10 µg/µl) is a recombinant wild-typeStreptococcus pyogenesCas9 nuclease expressed with a C-terminal nuclear localization signal (NLS), provided in 50% glycerol. The Cas9 protein solution has been verified to be sterile and well-tolerated by mammalian cells when electroporated as a ribonucleoprotein complex (RNP) with a single guide RNA (sgRNA) for knockout experiments, or as an RNP with a donor repair template for knockin experiments.

Homology-directed repair at theAAVS1orCXCR4genes.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 theAAVS1gene, and a template containing both HindIII and BamHI restriction sites was inserted into theCXCR4gene. 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 theCXCR4gene 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.

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-itIn VitroTranscription 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.

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 (B2MorPD-1) or double knockouts (B2 m+PD-1orTRAC+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.

Knockout of B2 m in CD3+ Pan T cells.In this experiment, CD3+ Pan T cells were electroporated with Cas9/sgRNA RNPs in order to knockoutB2 m. 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.