Neon transfection kit

To generate plasmid-based single-guide RNA (sgRNA) constructs (Supplementary Table 4), the all-in-one CRISPR-Cas9 vector (pAll-Cas9.Ppuro; Academia Sinica, Taiwan) was used as the backbone for cloning. Transfection was performed with the Neon Transfection Kit (Thermo Fisher Scientific). In total, 3 × 10⁵ K562 cells were collected from each culture flask, washed twice with 1 × PBS, centrifuged at 200 g for 5 min, and incubated in 10 μl resuspension Buffer R. Viability of K562-edited cells under different electroporation conditions was examined at 1 and 7 days after electroporation with the trypan blue exclusion method. DNA extraction and H19 CRISPR cleavage detection via PCR and gel electrophoresis were further performed after puromycin selection.
For transfection into human PBMC-iPSCs, the Alt-R CRISPR-Cas9 system (IDT) was used to generate a LNC000093-deleted allele. A pair of custom sgRNAs targeting two genomic regions flanking LNC000093 (Supplementary Table 4) was designed with proprietary algorithms of IDT. The sgRNAs were mixed with Alt-R^® S.p. Cas9 Nuclease V3 (IDT) to form ribonucleoprotein (RNP) complexes, which were subsequently used to assemble transfection complexes by incubation with Lipofectamine RNAiMAX reagent (Invitrogen). In total, 3 × 10⁵ cells per well in 24-well plates were transfected with 10 nM RNP complex.

V3 Cas9 RNP and chemically modified sgRNAs were incubated at a molar ratio of 1:2 (45 pmol to 90 pmol) at room temperature for 15 minutes [59 (link)]. After complex formation, 10⁵ NK-92 cells were transfected using the 10 µL Neon Transfection kit (ThermoFisher Scientific, Waltham, MA, USA) with the following electroporation settings: 1.200 V, 20 ms, 1 pulse (Neon System, ThermoFisher Scientific, Waltham, MA, USA). Finally, cells were transferred to a 48 well-plate in fresh NK-92 complete medium.

In total, 1.5 × 10⁶ activated and expanded T cells (three days post-isolation for healthy donors’ cells; one day post-isolation for patients’ cells) were transfected with 5 µg of mRNA (PE2 or PE2-GFP) and pegRNA (3 µM) using the “Expanded T cell 3” program of the GTx^TM Transfection System and R-50×3 processing assemblies (MaxCyte, Rockville, MD, USA). After electroporation, cells were treated with corresponding mediums to recover and proliferate as previously stated (Section 2.9). PE2 or PE2-GFP mRNA (1 µg) and pegRNA (900 nM) were utilized to transfect 3 × 10⁵ K–562 cells using the Neon Electroporation System (Thermo Fisher Scientific, Waltham, MA USA) and a 10 µL Neon transfection kit (Thermo Fisher Scientific, Waltham, MA USA) using the following electroporation settings: 1450 V, 10 ms, 3 pulses. On day 2 after transfection, gDNA was isolated from K–562 and T cells to analyze the editing efficiency.

To restore WASp expression by CRISPR/Cas9-mediated site-specific integration of a WAS cDNA, patient-derived WAS HSPCs (Figure 1—source data 2) were manipulated as described in Rai et al., 2020 (link). Briefly, 0.2 × 10⁶ cells were electroporated using a Neon Transfection kit (ThermoFisher Scientific) and re-suspended in ribonucleoprotein (RNP) complex. The RNP was made by incubating a gRNA targeting the first exon of WAS and High Fidelity Cas9 protein (Integrative DNA Technologies) at a molar ratio of 1:2 at 37°C for 15 min. The conditions for electroporation were 1600V, 10 ms, and 3 pulses. Following electroporation, cells were seeded at concentration of 1 × 10⁶ cells per ml and incubated at 37°C for 15 min after which an adeno-associated virus 6 (AAV6) donor vector containing a WAS cDNA flanked by WAS homology arms was added at 50,000 MOI (vector genomes/cell) for 12 hr. After that, cells were washed twice with PBS to remove viral particles and cultured in monocyte differentiation media.

After 2 days in culture, CD34+ HSPCs were electroporated using Neon Transfection kit (ThermoFisher Scientific). Briefly, 0.2 × 10⁶ cells were centrifuged and re-suspended in ribonucleoprotein (RNP) complex. The RNP was made by incubating a chemically modified (with 2-O-methyl-3′-phosphorothioate at the three terminal positions at both 5′ and -3′ ends; Synthego, USA⁵⁰ (link)) single gRNA targeting WAS,³ (link) with the Alt-R S.p. HiFi Cas9 protein⁵¹ (link) (Integrated DNA Technologies, USA) at a molar ratio of 1.5:1 at 37°C for 15 min. The condition for electroporation was 1600 V, 10 m, three pulses. Following electroporation, cells were seeded at concentration of 1 × 10⁶ cells per mL and incubated at 37°C for 15 min after which AAV6 at 25,000 MOI (Vector genomes/cell) was added and incubated.

Neurons were transfected by electroporation in serum and antibiotic-free medium according to the manufacturer’s instructions using a microporator (Thermofisher) and neon transfection kit (Thermofisher). The conditions optimal for cortical neuron transfection were determined to be 1500V, 10msec, 3 pulses. Transfected cells were plated onto coverslips coated with poly-L-lysine, laminin (10μg/ml) and CSA (50μg/ml), then cultured overnight in serum/antibiotic-free medium. The next day the medium was replaced with supplemented neurobasal medium as described above. 48h post-transfection the neurons were fixed and co-stained for the mcherry tag and β-tubulin as described below.
Neu7, SCTM41, COS7 and NG-108 cells were a gift from Professor James Fawcett. SH-SY5Y cells were a gift from Professor Jenifer Morton, both from the University of Cambridge.

siRNAs for mouse Rnf20, Gabpa, human RNF20, and the negative control were produced by Bioneer, Inc. (Daejeon, South Korea). Preadipocytes or differentiated adipocytes were mixed with siRNA or vectors using the Neon™ Transfection Kit (MPK1096 and MPK10096, Thermo Fisher) and transfected with a single pulse at 1100 V for 30 ms using a Microporator MP-100 (Digital Bio, Seoul, Korea). HEK293T cells were transfected using Lipofectamine™ RNAiMAX (13778075, Thermo Fisher) according to the manufacturer’s protocol or the calcium phosphate method. The sequence information of the siRNAs is described in Supplementary Data 1.