Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. This study demonstrates that chemical modifications to single guide RNAs (sgRNAs) significantly improve genome editing efficiency in human primary T cells and CD34⁺ hematopoietic stem and progenitor cells (HSPCs). The CRISPR-Cas system relies on sgRNAs to direct DNA cleavage by the Cas9 nuclease. The study shows that delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient and non-toxic method for CRISPR-Cas delivery, offering a simple and effective approach for genome editing. The sgRNA is composed of two RNAs, CRISPR RNA (crRNA) and trans-activating crRNA, which can be combined into a chimeric sgRNA. sgRNAs are typically about 100 nucleotides long, with 20 nucleotides at the 5' end hybridizing to a target DNA sequence. The remaining 80 nucleotides at the 3' end are critical for Cas9 recognition. The study tested various chemical modifications, including 2'-O-methyl (M), 2'-O-methyl 3' phosphorothioate (MS), and 2'-O-methyl 3' thioPACE (MSP), which were found to enhance sgRNA stability and efficiency. The study evaluated the effectiveness of chemically modified sgRNAs in targeting three human genes: IL2RG, HBB, and CCR5. The results showed that modified sgRNAs significantly increased indel frequencies and homologous recombination (HR) efficiency compared to unmodified sgRNAs. The modified sgRNAs also showed improved specificity, with higher on-target:off-target ratios. The study also tested the performance of chemically modified sgRNAs in primary cells, including stimulated and unstimulated T cells and CD34⁺ HSPCs. The results showed that modified sgRNAs were more effective in these cells, with higher indel frequencies and stable modification over time. The study also demonstrated that chemically modified sgRNAs could be delivered as RNA or ribonucleoprotein (RNP) complexes, which are more efficient and less toxic than DNA-based delivery methods. The study concludes that chemically modified sgRNAs offer significant advantages over unmodified sgRNAs in terms of genome editing efficiency, specificity, and compatibility with primary cells. These findings suggest that chemically modified sgRNAs have the potential to substantially improve a wide range of CRISPR/Cas applications in biotechnology and therapy.Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. This study demonstrates that chemical modifications to single guide RNAs (sgRNAs) significantly improve genome editing efficiency in human primary T cells and CD34⁺ hematopoietic stem and progenitor cells (HSPCs). The CRISPR-Cas system relies on sgRNAs to direct DNA cleavage by the Cas9 nuclease. The study shows that delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient and non-toxic method for CRISPR-Cas delivery, offering a simple and effective approach for genome editing. The sgRNA is composed of two RNAs, CRISPR RNA (crRNA) and trans-activating crRNA, which can be combined into a chimeric sgRNA. sgRNAs are typically about 100 nucleotides long, with 20 nucleotides at the 5' end hybridizing to a target DNA sequence. The remaining 80 nucleotides at the 3' end are critical for Cas9 recognition. The study tested various chemical modifications, including 2'-O-methyl (M), 2'-O-methyl 3' phosphorothioate (MS), and 2'-O-methyl 3' thioPACE (MSP), which were found to enhance sgRNA stability and efficiency. The study evaluated the effectiveness of chemically modified sgRNAs in targeting three human genes: IL2RG, HBB, and CCR5. The results showed that modified sgRNAs significantly increased indel frequencies and homologous recombination (HR) efficiency compared to unmodified sgRNAs. The modified sgRNAs also showed improved specificity, with higher on-target:off-target ratios. The study also tested the performance of chemically modified sgRNAs in primary cells, including stimulated and unstimulated T cells and CD34⁺ HSPCs. The results showed that modified sgRNAs were more effective in these cells, with higher indel frequencies and stable modification over time. The study also demonstrated that chemically modified sgRNAs could be delivered as RNA or ribonucleoprotein (RNP) complexes, which are more efficient and less toxic than DNA-based delivery methods. The study concludes that chemically modified sgRNAs offer significant advantages over unmodified sgRNAs in terms of genome editing efficiency, specificity, and compatibility with primary cells. These findings suggest that chemically modified sgRNAs have the potential to substantially improve a wide range of CRISPR/Cas applications in biotechnology and therapy.