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CRISPR-Cas9 systems enable innovative applications in mammalian cell engineering.However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain as limiting steps.Here we improved methods for gRNA synthesis and for delivery of Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) into a variety of mammalian cells through Lipofectamine(R) CRISPRMAXTM-mediated transfection or electroporation.Using these methods, the genome modification efficiencies were up to 94% in Jurkat T cells and 87% in induced pluripotent stem cells (iPSC) for a single target.For multigene targeting in Jurkat cells, two-locus and three-locus indels were achieved in approximately 93% and 65% of the resulting isolated cell lines, respectively.Further, the off-target cleavage rate was reduced using Cas9 protein when compared to plasmid DNA transfection.Taken together, we present a streamlined cell engineering workflow that enables gRNA design to analysis of edited cells in less than four days, resulting in highly efficient genome modulation in hard-to-transfect cells.The method is adaptable to high throughput, multiplexed genome-wide cell engineering.