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Background  CRISPR/Cas9 mediated gene knockout is a powerful tool for genome editing with the ability to target multiple genes simultaneously. Establishing an efficient, multiplexed gene knockout system using CRISPR/Cas9 that is both simple and robust in its application would further advance the adoption of CRISPR/Cas9 for genetic studies.    Results  In this study, we present a simple, versatile and highly efficient method to achieve acute gene knockout with CRISPR/Cas9 using chemically synthesized crRNA and tracrRNA oligos. We demonstrate that co-transfection of the crRNA:tracrRNA duplex into Cas9-expressing cells leads to target gene mutation and loss of target protein expression in the majority of the cell population. We also show that delivering three crRNAs targeting EGFP, KRAS and PTEN in the same reaction leads to the simultaneous knockout of all three genes. Direct comparison of multiplexed gene targeting by crRNA:tracrRNA and by siRNA indicates that these two methods are comparable in their efficiency and kinetics of gene silencing.    Conclusions  Our method is a convenient yet powerful tool to enable rapid and scalable gene knockout using CRISPR/Cas9 in mammalian cells.    Electronic supplementary material  The online version of this article (10.1186/s13578-019-0304-0) contains supplementary material, which is available to authorized users.

Multiplexed CRISPR/Cas9 gene knockout with simple crRNA:tracrRNA co-transfection