Pcmv t7 spry p2a egfp

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-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.