Cas9 nuclease v3

To generate SETD2 KO cell lines (HBL1, OCI-Ly7, SU-DHL-4, SU-DHL-2), parental cells were transduced with lentivirus expressing doxycycline inducible Cas9 and blasticidin resistance gene (Addgene, #83481) followed by 5 days of blasticidin selection. Cas9 expressing cells were transduced with lentivirus expressing a SETD2 specific sgRNA (SETD2G#1: 5’ AAA GAA ACA ATA GTA GAA GT 3’; SETD2G#2: 5’ AAT CTG ATG AAG ATT CTG TA 3’) and GFP (Addgene, #57822). 4 days post doxycycline induction, GFP⁺ cells were single cell sorted into 96 well plates and allowed to grow for at least two weeks. For RIVA, parental cells were electroporated with Amaxa Nucleofector Unit and the SF Cell line 4D-Nucleofector X kit (Lonza, PBC2–22500) to incorporate a recombinant Cas9 nuclease (Alt-R S.p. Cas9 Nuclease V3, Integrated DNA Technologies, 1081058), a SETD2 targeting Alt-R CRISPR-Cas9 crRNA (SETD2G#1: 5’ AAA GAA ACA ATA GTA GAA GT 3’; SETD2G#2: 5’ AAT CTG ATG AAG ATT CTG TA 3’ Integrated DNA Technologies) and Alt-R CRISPR-Cas9 tracrRNA (Integrated DNA Technologies, 1075927) using manufacturer’s protocol. Forty-eight hours after electroporation, ATTO550+ single cells were sorted into 96 well plates and allowed to grow for at least two weeks. Clones were screened by PCR amplification of a 500bp region encompassing the CRISPR-Cas9 cleavage site and verified by sanger sequencing at GENEWIZ.

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).