This study investigates the enhancement of CRISPR-Cas9 genome editing efficiency in human primary cells by chemically modified single guide RNAs (sgRNAs). The researchers synthesized sgRNAs with different chemical modifications at their termini and evaluated their performance in human cell lines and primary cells. They found that chemically modified sgRNAs, particularly those with 2′-O-methyl 3′-thioPACE (MSP) modifications, significantly improved genome editing efficiency in human primary T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). The modified sgRNAs enhanced both non-homologous end-joining (NHEJ)-mediated indel formation and homologous recombination (HR)-mediated gene disruption. The study also demonstrated that co-delivering Cas9 mRNA or protein with the modified sgRNAs further increased editing efficiency, suggesting a simple and effective RNA- or ribonucleoprotein (RNP)-based delivery method for CRISPR-Cas9 genome editing. The findings highlight the potential of chemically modified sgRNAs to enhance the utility of CRISPR-Cas9 technology in various biotechnological and therapeutic applications.This study investigates the enhancement of CRISPR-Cas9 genome editing efficiency in human primary cells by chemically modified single guide RNAs (sgRNAs). The researchers synthesized sgRNAs with different chemical modifications at their termini and evaluated their performance in human cell lines and primary cells. They found that chemically modified sgRNAs, particularly those with 2′-O-methyl 3′-thioPACE (MSP) modifications, significantly improved genome editing efficiency in human primary T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). The modified sgRNAs enhanced both non-homologous end-joining (NHEJ)-mediated indel formation and homologous recombination (HR)-mediated gene disruption. The study also demonstrated that co-delivering Cas9 mRNA or protein with the modified sgRNAs further increased editing efficiency, suggesting a simple and effective RNA- or ribonucleoprotein (RNP)-based delivery method for CRISPR-Cas9 genome editing. The findings highlight the potential of chemically modified sgRNAs to enhance the utility of CRISPR-Cas9 technology in various biotechnological and therapeutic applications.