Pcmv abemax p2a gfp

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 start codon of the TMUB1 gene was mutagenized with an adenine base editor to convert the ATG into GTG (Gaudelli et al, 2017 (link)). The gRNA was designed using the rgenome webtool (http://www.rgenome.net/), and primers were purchased from Sigma-Aldrich and cloned into gRNA Cloning Vector, a gift from George Church (plasmid # 41824; Addgene) (Mali et al, 2013 (link)) after digestion with AflII and through the Gibson assembly of the PCR product of the primer pair (guide sequence sense strand 5′-TTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCGCCATGACCCTGATTGAAG-3′, guide sequence antisense strand 5′-GACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACCTTCAATCAGGGTCATGGCGC-3′). The gRNA-containing vector was cotransfected with the pCMV_ABEmax_P2A_GFP, a gift from David Liu (plasmid # 112101; Addgene) (Koblan et al, 2018 (link)), and after 24 h sorted using INFLUX Cell-Sorter (BD Biosciences). Two 96-well plates were prepared, and the successful mutagenesis was tested via Western blot analysis.

The sequences of the AAV constructs used in this work were generated by using pLV302 and pLV312.3 (Addgene plasmid nos.119943 and 119944) where regions of interest were exchanged using NEBuilder HiFi DNA Assembly Master Mix (NEB no. E2621). Amino acid sequences are listed in Supplementary Note 1. PCR was performed using Q5 High-Fidelity DNA Polymerase (New England Biolabs). pCMV_ABEmax_P2A_GFP was a gift from David Liu (Addgene plasmid no. 112101). lentiGuide-Puro was a gift from Feng Zhang (Addgene plasmid no. 52963). The coding sequence of ABEmax was cloned into the mRNA production plasmid behind a T7 promoter for mRNA production and into pET His6 LIC cloning vector (2Bc-T, Addgene plasmid no. 37236) for protein production.