Guide-it Recombinant Cas9 (Electroporation-Ready)

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.

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.

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.