CRISPR Editing of CD34+ HSPCs

Chemically modified sgRNAs used to edit CD34⁺ HSPCs at either HBA2 or HBA1 were purchased from Synthego and TriLink BioTechnologies, and were purified by HPLC. The sgRNA modifications added were 2′-O-methyl-3′-phosphorothioate at the three terminal nucleotides of the 5′ and 3′ ends, as described previously^{30 (link)}. The target sequences for sgRNAs were as follows: sg1: 5′-CTACCGAGGCTCCAGCTTAA-3′; sg2: 5′-GGCAGGAGGAACGGCTACCG-3′; sg3: 5′-GGGGAGGAGGGCCCGTTGGG-3′; sg4: 5′-CCACCGAGGCTCCAGCTTAA-3′; and sg5: 5′-GGCAAGAAGCATGGCCACCG-3′. All Cas9 protein (Alt-R S.p. Cas9 Nuclease V3) used was purchased from Integrated DNA Technologies. RNPs were complexed at a Cas9/sgRNA molar ratio of 1:2.5 at 25 °C for 10 min before electroporation. CD34⁺ cells were resuspended in P3 buffer (Lonza) with complexed RNPs and electroporated using the Lonza 4D Nucleofector (program DZ-100). Cells were plated at 2.5 × 10⁵ ml⁻¹ following electroporation in the cytokine-supplemented media described previously. Immediately following electroporation, AAV6 was supplied to the cells at between 5 × 10³ and 1 × 10⁴ vector genomes per cell, based on titers determined using a Bio-Rad QX200 ddPCR machine and QuantaSoft software (v.1.7; Bio-Rad).