CRISPR/Cas9 gene editing can be performed using a variety of different delivery methods, including plasmid or mRNA transfection, viral transduction, or direct electroporation of recombinant Cas9-sgRNA ribonucleoprotein (RNP) complexes. We have developed a high-quality, recombinant Cas9 protein, Guide-it Recombinant Cas9, purified from E. coli, that is ready for use in electroporation protocols. Using sgRNAs produced with our Guide-it In Vitro Transcription Kit, we consistently achieve high levels of functional gene knockouts and homology-directed repair (HDR) in hard-to-edit cells, including hematopoietic stem cells (HSCs) and human iPS cells (hiPSCs).
Tech Note
Electroporation of Cas9-sgRNA ribonucleoproteins (RNPs) for gene editing in diverse cell types
Introduction
Results
Gene knockouts
To test the efficacy of our Cas9 protein, we designed sgRNAs against several common editing targets (CCR5, C4BPB, CYP2E1, and CTLA4), transcribed the guides using our Guide-it In Vitro Transcription Kit, and then electroporated sgRNA-Cas9 RNPs into the Cellartis human iPS cell line hiPSC-18. For comparison, we also delivered RNPs, including Cas9 protein, produced using other vendors' complete systems. Gene knockout was assessed using the Guide-it Mutation Detection Kit two days after electroporation (Figure 1). 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 RNPs produced using other vendors' recommended methods for guide RNAs.
Figure 1. 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. Numbers at the bottom of each gel represent gene editing efficiencies (expressed as a %) for the indicated RNPs.
To further test the gene editing efficiency, knockout of the CD81 gene was performed in two induced pluripotent stem cell lines, hiPSC-18 and hiPSC-22, using Cas9-sgRNA electroporation. The knockout efficiency 10 days after electroporation was 91% in hiPS-18 cells and 85% in hiPSC-22 cells (Figure 2).
Figure 2. Knockout of CD81 in induced pluripotent stem cells. In this experiment, two induced pluripotent stem cell lines, hiPSC-18 and hiPSC-22, were electroporated with Cas9-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.
Gene kockin via HDR in CD34+ HSCs
As a second test of our recombinant Cas9, we electroporated repair templates containing a HindIII restriction site for AAVS1 and HindIII and BamHI sites for CXCR4 in combination with our Cas9-sgRNA RNPs and assayed for gene editing by performing restriction digests. These experiments were done in CD34+ hematopoietic stem cells (HSCs). The experimental design and results are shown in Figure 3.
Figure 3. Homology-directed repair at the AAVS1 or CXCR4 genes. Panel A. These diagrams demonstrate how the HDR experiments in CD34+ HSCs were done. An 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 in length 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.
Conclusions
Guide-it Recombinant Cas9 is a pure and effective protein ready for use in any gene editing experiment. 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. In our experiments, a knockout efficiency of 85–91% could be obtained 10 days postelectroporation in hiPSC cell lines.
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