The discovery of the CRISPR-Cas system has revolutionized the biomedical research due to its versatility and ease of use. It offers extraordinary potential for developing novel cancer immunotherapeutic approaches. Recently, immune checkpoint blockade (ICB) has become one of the most promising strategies in cancer immunotherapies by blocking down-regulators of immunity and reverting tumor-induced immune suppression. However, only a portion of patients can respond to this treatment. To overcome this limitation, a folate receptor-specific Cas9 ribonucleoprotein (RNP) nanocarrier was developed for the effective intracellular transport of Cas9 RNP and PD-L1 (CD274)/PVR (CD155) dual immune checkpoint disruption. The nanocarrier is based on a hydroxystearic acid-containing lipopeptide and further conjugated with folic acid (FolA) and polyethylene glycol (PEG) for specific delivery of RNP in folate receptor α overexpressing cancer cells. In vitro studies showed that FolA-modified nanocarriers significantly enhanced the knockout of PD-L1 and PVR genes in colon carcinoma CT26 cells compared to PEG- and unmodified nanocarriers. Moreover, the FolA-modified nanocarriers achieved ~25% PD-L1/PVR dual gene disruption and totally ~40% gene editing efficiency in CT26-bearing mice. We expect that the established receptor-specific Cas9 RNP delivery system can provide a promising approach to increase the efficacy of cancer immunotherapy by combined immune checkpoints disruption.