Ng abemax

Human codon-optimized base editing constructs were a kind gift from David Liu; pCMV_ABEmax_P2A_GFP (plasmid #112101; Addgene), pCMV_AncBE4max_P2A_GFP (plasmid#112100; Addgene). pCMV_SpCas9-NG_ABEmax_P2A_GFP, pCMV_SpCas9-NG_AncBE4max_P2A_GFP and pCMV_SaKKH_AncBE4max_P2A_GFP were constructed by PCR amplification (Q5, NEB) amplifying everything except for SpCas9 using pCMV_ABEmax_P2A_GFP and pCMV_AncBE4max_P2A_GFP. Coding sequences for SpCas9-NG and SaKKH were PCR amplified using the following plasmids NG-ABEmax (plasmid #124163; Addgene) and SaKKH-ABEmax (Plasmid #119815; Addgene) that were a kind gift from David Liu. Coding sequences and plasmid backbones were combined using the NEBbuilder HiFi DNA assembly mastermix (NEB) and subsequently transformed using OneShot Mach1t1 (Thermo Fisher Scientific) cells and plasmid identity was checked by Sanger sequencing (Macrogen). The empty sgRNA plasmid backbone for SpCas9 and its derivatives was a kind gift from Keith Joung (BPK1520, Addgene plasmid #65777). Spacer sequences targeting all genes in this study were cloned in the sgRNA plasmid backbone using inverse PCR (Q5 NEB) and subsequently transformed using OneShot Mach1t1 (Thermo Fisher Scientific) cells and plasmid identity was checked by Sanger sequencing (Macrogen). Primer sequences for sgRNA generation can be found in Supplementary Table 3.

The original plasmid NG-ABEmax expressing the TadA-TadA*-nSpCas9-NG fusion protein was obtained from Addgene (Addgene plasmid #124163). The plasmid pSin-EGFP containing an EGFP gene, IRES and Puromycin genes were generated was generated from pSIN-EF2-Lin28-Puro(obtained from Addgene; #16580) using EcoR I and BamH I restriction enzyme sites^{22 (link)}. The sgRNA expression cassettes were generated by standard protocol. Desired point mutations were introduced into the coding sequence of EGFP by PCR to generate EGFP-variant, dEGFP1, and dEGFP2. All plasmids were confirmed by Sanger sequencing. The oligonucleotide sequences used for plasmid construction in this study are listed in Supplementary Tables 4–9.

NIH3T3-RPE65 (rd12) cells were seeded on a 24-well plate 18 h prior to transfection. At ~70% confluency, cells were transfected with 750 ng of ABE-expression plasmid and 250 ng of sgRNA-expression plasmid using 1.5 µl of Lipofectamine 3000 (Thermo Fisher, no. L3000001) per well. Four kinds of ABE-expression plasmids were used: pCMV-ABEmax (Addgene plasmid #112095), NG-ABEmax (Addgene plasmid #124163), xABEmax (Addgene plasmid #119813) and pCMV-ABEmax-NRRH^{23 (link)}. Two sgRNA-expression plasmids were generated as previously described^{15 (link)}. Cells were harvested for genomic DNA purification 48 h post-transfection.

Cloning of pegRNA plasmids was performed according to previously described protocols^{4 (link)}. In brief, the pU6-pegRNA-GG-Vector (Addgene #132777) was digested overnight with BsaI-HFv2 (NEB) and the 2.2 kb fragment was isolated. Oligonucleotide duplexes containing the desired pegRNA spacer, pegRNA extension, and pegRNA scaffold sequences were ordered with the appropriate overhangs and annealed. Annealed pegRNA duplexes were cloned into the pU6-pegRNA-GG-Vector using Golden Gate assembly with BsaI-HFv2 (NEB) and T4 DNA ligase (NEB) in a protocol of 12 cycles of 5 min at 16 °C and 5 min at 37 °C. For cloning of sgRNAs used for PE3 and ABE-NG, we replaced the BsmBI restriction sites of the sgRNA expression vector BPK1520 by BbsI restriction sites using PCR, which allowed direct ligation of sgRNA-spacer duplexes^{21 (link)}. All pegRNA, sgRNA, HDR template, and primers sequences used in this work are listed in Supplementary Tables 1–3 and were synthesized by Integrated DNA Technologies. pCMV-PE2 (Addgene #132775), pU6-pegRNA-GG-acceptor (Addgene #132777), and NG-ABEmax (Addgene #124163) were gifts from David Liu; BPK1520 (Addgene #65777) was a gift from Keith Joung; pSpCas9(BB)-2A-Puro (PX459, Addgene #62988) was a gift from Feng Zhang.