Fig. 5: Iterative genome editing with CRISPR SWAPnDROP.
From: A multifunctional system for genome editing and large-scale interspecies gene transfer
a The use of the two pSwap plasmids (CmR/GmR) allows iterative genome editing. Each harbouring either the I-SceI or I-CreI meganuclease genes and the opposite recognition site I-CreI site or I-SceI site, respectively. During the editing, the meganuclease genes are removed leaving the pSwap plasmid only with the opposite recognition site. Subsequent transformation of the other pSwap plasmid leads to the elimination of the previous plasmid and allows for another round of editing. Cell colours represent the corresponding editing step. b Next-generation sequencing of E. coli mutant after 12 consecutive edits. Shown is the next-generation sequencing (NGS) coverage of a E. coli mutant strain after 12 consecutive edits (light grey) compared to the wild-type E. coli MG1655 (dark grey). For the mutant strain, several recombination sites (FRT/loxP) as well as random DNA and origins of replication were inserted into the chromosome using iterative CRISPR SWAPnDROP genome editing. Mutant and wild-type E. coli NGS reads were aligned against the mutant reference genome and the sectors of each edited site (I–X) as well as the complete genome coverage (circle) are shown. Reads at all insertion locations (dashed lines) are present for the mutant strain, while no reads are present for the wild-type strain (red rectangle). (I) and (V) show the coverage of a chromosomal location after 2 different edits at the same position. For (I), the native origin of replication oriC of E. coli was first replaced with the F-plasmid derived oriS origin of replication. oriS was then replaced again with oriC while introducing scar sites flanking the oriC. Scar sites were introduced due to using HA, INS and HB plasmids instead of HAIB plasmid. The absence of those flanking scars in the wild-type is highlighted with red rectangles in (I). For (V), oriC was first inserted into the insPQ locus and then replaced with a random sequence. The absence of the random sequence in the wild-type strain is highlighted with a red square.
