To introduce DSBs in mammalian cells using an RNP complex, 2E+5 cells were transfected with Cas9 protein (4.5–45 µg) premixed with in vitro transcribed sgRNA (6–60 µg). Cas9 protein in storage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, and 10% glycerol) was mixed with sgRNA dissolved in nuclease-free water and incubated for 10 min at room temperature. No more than 4 µL of the RNP mixture was added to 20 mL of the Nucleofection solution. For plasmid-mediated expression of RGENs, 2E+5 cells were co-transfected with 1 mg of Cas9-encoding plasmid and 1 µg of sgRNA-expressing plasmid in K562 and BJ fibroblasts or 2.4 µg of Cas9-encoding plasmid and 1.6 µg of sgRNA-expressing plasmid in H9 hES cells. K562 cells were transfected with the Amaxa SF Cell Line 4D-Nucleofector Kit using Program FF-120 (Lonza), and H9 and BJ cells were transfected with the Amaxa P3 Primary Cell 4D Nucleofector Kit using Program CB-150 and DT-130, respectively, according to the manufacturer’s protocol. Cells were analyzed 2 days after transfection, unless indicated otherwise. Lonza summary: The authors delivered purified recombinant Cas9 protein and guide RNA, from CRISPR system, into K562, human fibroblasts and pluripotent stem cells using the 4D Nucleofector system. This reduces off-target mutations and was less stressful for human embryonic stem cells, in comparison to Cas9 plasmid transfection, due to the rapid degradation of the transfected Cas9 ribonucleoprotein after delivery. The mutation frequency reached a plateau one day upon Nucleofection when Cas9 was transfected as a protein versus three days when it was delivered as a plasmid. The versatility of Nucleofection for delivering different type of substrates including large proteins without further Nucleofection optimization enabled here the improvement of genome engineering methods.