Abe8.20 m

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 ABE8e, ABE8.17-m, ABE8.20-m, pCMV-ABE7.10, pCMV_ABEmax, and pCMV-T7-SpRY-P2A-EGFP plasmids were obtained from Addgene (#138489, #136298, #136300, #102919, #112095, and #139989, respectively). The construction of Nme2-ABEmax was described in detail in our previously published study.¹³ (link) The open reading frames of nNme2Cas9 and SpRYCas9 were amplified by PCR for subsequent assembly into a base-editing architecture backbone using a ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China). TadA8.17 DNA fragments were synthesized and cloned into nNme2-ABE8.17 and SpRY-ABE8.17 by GenScript Biotech (Nanjing, China). The D10A mutation was introduced into the SpRY-ABE8.17 plasmid. Site-directed mutagenesis was performed using a Fast Site-Directed Mutagenesis Kit (TIANGEN, Beijing, China). To construct the ABE8.17-NL plasmid, the reading frame encoding TadA-8.17 was amplified by PCR and used to replace TadA-8.17 and the linker fragment within ABE8.17-m, thus generating ABE8.17-NL. The sgRNA plasmid was constructed and inserted into the PUC57 and 74,707 vectors. Spacer oligos and sgRNA scaffold oligos were synthesized and cloned into the 74,707 and pUC57-sgRNA expression vectors.