Genome editing by the delivery of pre-assembled Cas9 ribonucleoproteins (Cas9 RNP) is an increasingly popular approach for cell types that are difficult to manipulate genetically by the conventional plasmid and viral methods. Cas9 RNP editing is robust, precise, capable of multiplexing, and free of genetic materials. Its transient presence in cells limits residual editing activity. This protocol describes the preparation of recombinant Streptococcus pyogenes Cas9 (SpCas9) protein by heterologous expression and purification from Escherichia coli, and the synthesis of CRISPR guide RNA by in vitro transcription and PAGE purification. SpCas9 is the first CRISPR Cas9 discovered (Jinek et al., 2012) and is also one of the most characterized Cas enzymes for genome editing applications. Using this formulation of Cas9 RNP, we have demonstrated highly efficient genome editing in primary human T and natural killer (NK) cells by electroporation, and in fungi and plants by polyethylene glycol-mediated transformation. Our protocol of Cas9 RNP preparation is consistent and straightforward to adopt for genome editing in other cell types and organisms.