Clonexpress multis

The pTrcHisB was used as the plasmid backbone. The genes, luxS, mtn, lsrACDB, were PCR-amplified from the E. coli MG 1655 genome and ligated to pTrcHisB (KpnI, EcoRI, BamHI, SacI, BglII were used in this process) using ClonExpress II (Vazyme, China), to form three plasmids: pLuxS, pLuxS-Mtn, pLsrACDB. Then the PstI and BstBI digested lsrK segment was inserted into the same restriction sites of the pLsrACDB vector and transformed into E.coli K12, resulting in pLsrACDBK. The construction of plasmids pLsrACDBFG and pLsrACDBFGK was analogous to pLsrACDBK construction. For the construction of pLGFP, and the promoter sequence of lsrR, lsr and the opening reading frame (ORF) of green fluorescent protein (GFP) were PCR-amplified. This two fragments were ligated in pTrcHisB (BamHI, SacI were used in this process) using ClonExpress MultiS (Vazyme, China). Primers used in this study are listed in Additional file 1: Table S1. Procedures of cloning, DNA purification, and transformations were performed using standard protocols. All plasmids were confirmed by restriction enzyme digestion and DNA sequencing.

The multiplex sgRNAs were fused by overlapping PCR to obtain 3 sgRNA tandem structures, gtRNA–gag, gtRNA–pol1 and gtRNA–pol2. The primers are listed in Supplementary Table S1. The PCR products were then cloned into the BbsI-digested U6–sgRNA cloning vector to obtain the sgRNA-expressing plasmids pU6–gtRNA–gag, pU6–gtRNA–pol1, and pU6–gtRNA–pol2. The sequence of the seven tandem sgRNAs: U6–gtRNA–gag, U6–gtRNA–pol1 and U6–gtRNA–pol2 are listed in Supplementary Table S2. Then, the 7 sgRNA expression elements were introduced into the MluI-cleaved pCMV-BE4 max-P2 A-Puro vector using recombination kit (ClonExpress^® MultiS, Vazyme, Gaithersburg, MD, USA) to obtain the pCMV-BE4 max-P2 A-Puro-tandem sgRNA plasmid. Finally, all expressed elements such as the 7 sgRNAs, BE4 max and the puromycin resistance gene were cloned into the middle of pCEP4 by HindIII and PmeI to obtain the pCEP4-BE4 max-P2 A-Puro-tandem sgRNAs, which were also named MAIO-epiCBE. The procedure for the construction of the MAIO-epiCBE plasmid is shown in Supplementary Figure S1.

Plasmids expressing Target-AID (pcDNA3.1-Target-AID) and ABE7.10 (pcDNA3.1-ABE7.10) were obtained from our lab. The pcDNA3.1-ACBE expressing plasmid was constructed by cloning the PmCDA1 and UGI fragments amplified from pcDNA-Target-AID into the C terminus of plasmid pcDNA3.1-ABE7.10. The pcDNA3.1-ACBE-0C and pcDNA3.1-ACBE-20C were constructed similarly to pCDNA3.1-ACBE but with the 0 and 20 aa linkers, respectively. pcDNA3.1-ACBE-16N and pCDNA3.1-48 N were constructed by fusing the heterodimer of TadA with 16 and 48 aa linkers, respectively, into the N terminus of pcDNA3.1-Target-AID. These vectors were generated by using a recombination kit (ClonExpress® MultiS, Vazyme). All sgRNAs used in this study were designed in accordance with the G-N19-NGG rule. In brief, two complementary oligonucleotides of sgRNAs were synthesized and then annealed to double-stranded DNAs. The annealed products were then cloned into the BbsI-digested U6-sgRNA cloning vector, and the sgRNA-expressing plasmids were obtained. All newly constructed plasmids were confirmed by Sanger sequencing.