Fig. 6: Transfer and integration of a 151 kb chromosomal region via CRISPR SWAPnDROP.
From: A multifunctional system for genome editing and large-scale interspecies gene transfer
a Scheme of the deletion of the 167 kb del4 chromosomal region in E. coli MG1655 (top). Swap and conjugational transfer of the 151 kb MFDdel4 chromosomal region from E. coli MFDpir to E. coli MG1655 Δdel4 and subsequent integration (bottom). Cell colours represent the corresponding editing step. b Shown are agarose gels of PCRs from the integration of a 151 kb chromosomal region in E. coli. PCRs were done for E. coli MG1655 (acceptor precursor), the acceptor (E. coli MG1655 Δdel4) and the edited strain (E. coli MG1655 Δdel4::MFDdel4). PCRs “1–6” result in the amplification of fragments of the del4 region evenly distributed over the 167 kb (E. coli MG1655) and 151 kb (E. coli MFDpir), respectively. PCR “7” result in the amplification of a 3000 bp fragment, if the del4 region is missing. PCR “8” and “9” amplify the transition of the del4 region to the adjacent chromosomal regions. Upon integration of the 151 kb MFDdel4 region into the E. coli MG1655 Δdel4 chromosome, small regions flanking the 151 kb region were deleted resulting in DNA bands of lower size compared to the wild-type chromosome. This is additionally highlighted with the colours green and red. Dashed line indicates the band size of a 2000 bp DNA fragment for a better comparison. PCR was repeated once with identical results. Chromosomal overviews including homologous regions (HA, HB, Hα, Hβ of each PCR are shown above each agarose gel. c Shown is the next-generation sequencing (NGS) coverage (reads/base) of E. coli MG1655 Δdel4::MFDdel4 (light grey) and E. coli MG1655 (dark grey) mapped against the E. coli MG1655 reference genome. The middle plot shows the coverage of the del4 region. For E. coli MG1655 Δdel4::MFDdel4, reads for the 16 kb deletion, only present in the integrated MFDdel4 region, are missing (red rectangle). The upper and lower plots showing the detailed coverage of the del4 flanking regions. Upon integration of the MFDdel4 region, small flanking regions were deleted, shown as missing reads between the dashed lines.
