Cancer gene therapy based on the clustered regularly interspaced short palindromic repeat (CRISPR) system has been challenging due to the poor delivery and efficacy in vivo. Herein, we report the development of nanoassembled ribonucleoprotein complexes (NanoRNP), which can efficiently block the PD-L1 immune checkpoint and induce an antitumor effect in vivo. We utilize CRISPR-associated protein 9 (Cas9) that is chemically derivatized with a low-molecular weight polymer, which condenses with single-guide RNA and modified DNA oligonucleotides to form stabilized NanoRNPs. Delivery of the NanoRNP into B16 melanoma cells in vitro leads to efficient internalization and gene disruption with low cytotoxicity, leading to sustained downregulation of PD-L1. In vivo delivery in a mouse melanoma model demonstrates that the NanoRNP can induce indels in the target cells of the tumor at high frequencies, resulting in major suppression of tumor growth, without involving combinatorial treatment. Blockade of the PD-L1 checkpoint by the NanoRNP in tumor tissues promotes T cell infiltration and effector cytokine release, which are characteristics of the activation of anti-tumor immunity, and inhibition of immunosuppressive myeloid cells. The current system suggests a promising strategy as an in vivo gene editing platform for cancer immunotherapy.