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 (Electroporation-Ready), 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).
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Gene editing technical notes
- Screening for effective guide RNAs
- Electroporation-grade Cas9 for editing in diverse cell types
- Site-specific gene knockins using long ssDNA
- Mutation detection kit comparison
- sgRNA-Cas9 delivery to many cell types
- Cas9 Gesicles—reduced off-target effects
- CRISPR/Cas9 gene editing with AAV
- Tet-inducible Cas9 for gene editing
- Monoallelic versus biallelic mutants
- Indel identification kit for mutation characterization
- Fast Cre delivery with gesicle technology
- Phenotypic screen using sgRNA library system
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Electroporation of Cas9-sgRNA ribonucleoproteins (RNPs) for gene editing in diverse cell types
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.
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).
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.
Guide-it Recombinant Cas9 (Electroporation-Ready) 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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