Agarose gel purification

Clinical isolates were screened for the presence of z4861 and yegN by the polymerase chain reaction (PCR). Primers covering the conserved regions among different E. coli strains were used (Additional file 1: Table S2). Representative PCR products were confirmed by sequencing after agarose gel purification (Qiagen, Germany).

Plasmid-borne SpCas12f1 and AsCas12f1 CRISPR systems described earlier^{1 (link)} were engineered to also encode a 10×His:MBP tag fused to N-terminus of the cas12f1 gene. Additionally, a sequence encoding the tobacco etch virus (TEV) protease recognition sequence (ENLYFQS) was also included. The SpCas12f1 plasmid was digested with EcoNI and NcoI restriction enzymes (NEB) and the backbone was isolated by agarose gel purification (Qiagen). Next, a synthesized DNA fragment (Genscript) containing a 5’ EcoNI restriction site, T7 promoter, lac operator, and ribozyme-binding sequence in addition to the sequence encoding the 10×His:MBP:TEV tag followed by an inverted BbsI site incorporating a sequence that upon digestion would yield a compatible NcoI overhang was digested with EcoNI and BbsI and column purified (Qiagen). The two purified fragments were then joined using T4 DNA ligase (NEB), transformed into One Shot TOP10 E. coli cells (Invitrogen), and constructs confirmed by Sanger sequencing. For AsCas12f1, a similar strategy was used except EcoNI and AvaI restriction enzymes (NEB) were used. Links to the plasmid sequences (pMBP-SpCas12f1 and pMBP-AsCas12f1) are provided in Supplementary Table 1.