Erythrocyte membrane-camouflaged nanodelivery strategy enhances gene editing efficiency of Cas9 RNP for boosting tumor senescence

CRISPR-Cas9 system has emerged as an effective tool for sequence-specific gene knockout through non-homologous end joining (NHEJ). However, the inefficient precise editing of genome sequences remains a challenge for clinical treatment. Herein, an erythrocyte membrane (EM)-camouflaged and tumor microenvironment (TME)-responsive nanosystem was constructed to achieve synergistic combination of enhanced Cas9 ribonucleoprotein (RNP) gene editing efficiency and telomere dysfunction, and thus precise induced tumor inhibition. The EM-camouflaged nanosystem escaped from the reticuloendothelial system (RES) and was endocytosed by tumor cells. Azidothymidine (AZT) was gradually released as the erythrocyte shell ablation, which competitively prevented the binding of single nucleotides to the telomere replication template TR, terminate the extension of the telomerase DNA chain. Additionally, Cas9 RNP was released through disulfide bond cleavage and entered into the nucleus to realize genome editing of telomerase reverse transcriptase (TERT) gene. Simultaneous, AZT enhanced NHEJ pathway to improve the gene editing efficiency of Cas9 RNP, which further promoted the progressive telomere erosion and thus induced tumor inhibition. Overall, this biomembrane-camouflaged nanosystem realizes enhanced Cas9 RNP gene editing efficiency with high-level biosafety and promotes tumor senescence by triggering telomere disorder, which provides strategy for improving the gene editing efficiency to induce senescence of tumor cells.