Nucleocuvette strip

Chemically modified gRNA oligomers were synthesized by Synthego (Redwood City, CA, USA). The 20-nt-long specific sequences for targeting the CD55 gene were as follows: 5′-GGCGCGCCATGACCGTCGCG-3′ and 5′- GTTGTGCCTGCCGGCCGTGT-3′. 40 pmol of the Cas9 2NLS nuclease (Synthego) was complexed with 2 gRNAs (50 pmol each) to form RNPs for 10 min at room temperature prior to electroporation. After that, 0.2 × 10⁶ BMI1-induced erythroblasts were reconstituted in P3 primary cell electroporation solution, according to the manufacturer’s instructions (Lonza, Basel, Switzerland) and mixed with RNP complexes. The supplemented cell solution was transferred into the Lonza 4D-Nucleofector with 20 μL Nucleocuvette strips and electroporated using the EO-100 program. Recovered cells were cultured for 72–96 h prior to flow cytometry analysis and erythroid terminal differentiation.

Synthetically modified sgRNA constructs were purchased from Synthego (refer to Supplementary Table S1 for sgRNA sequences). Ribonucleoprotein (RNP) assembly was performed by mixing 2 to 3 sgRNAs (a total of 120 pmol/L) with 8.5 μg recombinant Cas9 (Invitrogen A36499). The resulting RNP mix was electroporated into 0.3 × 10⁶ cells using a Lonza 4D Nucleofector, program DJ-100, in 20 μL Nucleocuvette strips (Lonza V4XC-2032). Unless otherwise noted, cells were incubated in media for 72 hours postelectroporation before subsequent analyses. Knockout efficiency was confirmed by Western blotting (Supplementary Fig. S4A). The following antibodies were used: actin (Santa Cruz sc-47778), IRF8 (Cell Signaling 5628s), MEF2C (Cell Signaling 5030s), MYB (Abcam ab45150). A guide RNA targeting the AAVS1 “safe harbor” locus was used as a negative control (44 (link)).

HT-29 cells were plated two days before transfection, to reach 70–80% confluency at the time of transfection. 0.5 × 10⁶ cells per transfection were collected for each condition. Cells were transfected with RNP complex and HDR templates by nucleofection with SF Cell Line 4D-Nucleofector X Kit (Lonza) using 20-ml Nucleocuvette Strips, as described by the manufacturer (Program FF137). Cells were immediately resuspended in 100 ul of culturing medium and plated into 1.5 ml of pre-warmed culturing medium in 24-well tissue culture plates. T7E1 assay, and site-specific PCR reactions were performed 72 h after nucleofection in order to check cleavage efficiency and integration, respectively. After confirming the correct integration and one-week regeneration cells were plated into 15 cm dish. The next day media was changed to antibiotic containing media (500 ug/ml neomycin and 20 ug/ul blasticidin) and antibiotic selection was performed. After an additional week, formed colonies were collected by trypsinization and resuspended in culture media. After regeneration culturing period (3–5 days), cell lines were tested by FACS analysis and site-specific PCR.