Abe8e

To generate SpRYc, the N-terminal ORF of Sc++ (Addgene Plasmid #155011), corresponding to residues (1–1119) was PCR amplified and assembled using Gibson Assembly into the pCMV-T7-SpRY-P2A-EGFP backbone (Addgene Plasmid #139989), preserving residues 1111–1368 of SpRY’s ORF. pCMV-T7-SpCas9-P2A-EGFP (Addgene Plasmid #139987) was used for SpCas9, and Sc++ was similarly integrated within the backbone. Analogously, the ORFs of SpCas9, SpRY, and SpRYc were integrated within the ABE8e (Addgene Plasmid #138489) and AncBE4Max (Addgene Plasmid #112094) backbones. sgRNA plasmids were constructed by annealing oligonucleotides coding for crRNA sequences (Table S1) as well as 4 bp overhangs, and subsequently performing a T4 DNA Ligase-mediated ligation reaction into a plasmid backbone immediately downstream of the human U6 promoter sequence. Assembled constructs were transformed into 50 μL NEB Turbo Competent E. coli cells, and plated onto LB agar supplemented with the appropriate antibiotic for subsequent sequence verification of colonies and plasmid purification.

The ABE8e, ABE8.17, AncBE4max, and PE plasmids were obtained from Addgene (#138,489, #136,298, #138,270 and #136,463). mCherry-T2A-GFP was described in detail in our previously published study [39 (link)]. The DNA fragments of deletion variants from ABE, CBE and PE were used for in-fusion cloning by ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China). All PCR primers are listed in Additional file 1: Table S2 and were synthesized by Sangon Biotech. The pegRNA and sgRNA were synthesized by Genscript Biotech (Nanjing).

All cell lines were infected with the construct library aiming at a multiplicity of infection (MOI) of 0.8 and a coverage of 800×. Each cell line was infected twice and treated as two biological replicates. To screen, 293FT cells were cultured in media containing 2 μg/ml puromycin for one week to select for infected cells. 6 × 10⁷ cells were then seeded into six tissue culture dishes with 150 mm diameter in 20 ml media. Twenty-four hours later, the media was refreshed with 15 ml of media. Transfection mixes were prepared in two steps (protocol adapted from (34 (link))). First, 16 ml of Opti-MEM was mixed with 72 μg of base editor encoding plasmid (BE4, Addgene #112673, ABE8e, Addgene #138489 or ABE8.20-m, Addgene #136300), 8 μg of pCS-GFP plasmid and 800 μl Plus reagent. Secondly, 16 ml Opti-MEM was mixed with 400 μl Lipofectamine 3000 (Invitrogen) and 1600 μl Lipofectamine LTX. The two solutions were mixed together, incubated for 30 min at room temperature and 3.2 ml of the transfection mix was transferred to each tissue culture plate. Forty-eight hours later, 15 ml of media was added to cells. After 14 h cells were harvested and a subsample of cells were used to check for transfection efficiency via flow cytometry. The data was analysed with FlowJo.

The rA1-CBE, eCDA1-CBE, dSaCas9 and ABE8e plasmids were obtained from Addgene (#112093, #122608, #138162 and #138489). The DNA fragments of Ade1-Ade7 were synthesized and cloned into the pcDNA3.1 vector (GenScript). The DNA fragments of A3A, A3B, A3Bctd, A3Gctd, A3Gntd, dA3G and miR-122BS were synthesized and cloned into the corresponding vectors (GenScript). Plasmid site-directed mutagenesis was performed using a Fast Site-Directed Mutagenesis Kit (Tiangen). The sequences of plasmids are listed in the Supplementary sequence.

To generate SpRYc, the N-terminal ORF of Sc + + (Addgene Plasmid #155011), corresponding to residues (1–1119) was PCR amplified and assembled using Gibson Assembly into the pCMV-T7-SpRY-P2A-EGFP backbone (Addgene Plasmid #139989), preserving residues 1111–1368 of SpRY’s ORF. pCMV-T7-SpCas9-P2A-EGFP (Addgene Plasmid #139987) was used for SpCas9, and Sc + + was similarly integrated within the backbone. Analogously, the ORFs of SpCas9, SpRY, and SpRYc were integrated within the ABE8e (Addgene Plasmid #138489), AncBE4Max (Addgene Plasmid #112094), or pCAG-CBE4max-SpRY-P2A-EGFP (Addgene Plasmid #139999) backbones, enforcing a D10A mutation. sgRNA plasmids were constructed by annealing oligonucleotides coding for crRNA sequences (Supplementary Fig. 1, Supplementary Table 1), as well as 4 bp overhangs, and subsequently performing a T4 DNA Ligase-mediated ligation reaction into a plasmid backbone immediately downstream of the human U6 promoter sequence. The MECP2 editing locus containing all common Rett syndrome mutations was synthesized as a gBlock from IDT and inserted via Gibson cloning to a lentiviral vector harboring puromycin resistance. Assembled constructs were transformed into 50 μL NEB Turbo Competent E. coli cells, and plated onto LB agar supplemented with the appropriate antibiotic for subsequent sequence verification of colonies and plasmid purification.