| Summary: | Genome editing using the CRISPR/Cas9 system is one of the trendiest methodologies in the scientific community. Many genome editing approaches require recombinant <i>Streptococcus pyogenes</i> Cas9 (<i>Sp</i>Cas9) at some point during their application, for instance, for in vitro validation of single guide RNAs (SgRNAs) or for the DNA-free editing of genes of interest. Hereby, we provide a simple and detailed expression and purification protocol for <i>Sp</i>Cas9 as a protein fused to GFP and MBP. This protocol improves protein yield and simplifies the purification process by overcoming the frequently occurring obstacles such as plasmid loss, inconsistent protein expression levels, or inadequate protein binding to affinity resins. On average, this protocol yields 10 to 30 mg of purified, active, His6−MBP−<i>Sp</i>Cas9 NLS−GFP protein. The purity addressed through SDS-PAGE is > 80%.
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