CRISPR-Cas9 Editing of CCR5 in HSPCs

An sgRNA targeting CCR5 exon 3 (sequence; 5ʹ-GCAGCATAGTGAGCCCAGAA-3ʹ) was purchased from TriLink Biotechnologies (San Diego, CA, USA) with the chemical modification 2′-O-methyl-3ʹ-phosphorothioate^{25 (link)}. Cas9 and Hifi Cas9 were purchased from Integrated DNA Technologies (IDT, San Jose, CA, USA Catalog #1081058 and #1081060). The editing procedure was performed as follows: sgRNA and Cas9 protein were complexed at a molar ration of 1:2.5 (sgRNA:Cas9) at room temperature for 5 min. The RNP was electroporated into human CD34+ HSPCs 48 h after thawing using the Lonza 4D nucleofector with the following conditions: pulse code: DZ100; cell density: 1 × 10⁶ cells in 100 µl; [Cas9]: 30 µg; [sgRNA]: 15 µg. Following electroporation, cells were immediately rescued with HSPC culture media pre-warmed to 37 °C. rAAV6 was applied to cells at a MOI of 10,000–20,000. The frequency of indel formation was quantified using Tracking Indels by Decomposition (TIDE)^{70 (link)}. CCR5 expression was quantified by flow cytometry using anti-human CCR5-APC antibody (BD Biosciences, #556903).