Analysis of RecA-independent recombination events between short direct repeats related to a genomic island and to a plasmid inEscherichia coliK12

Abstract

RecA-independent recombination events between short direct repeats, leading to deletion of the intervening sequences, were found to occur in two genetic models in the Escherichia coli K12 background. The first model was a small E. coli genomic island which had been shown to be mobile in its strain of origin and, when cloned, also in the E. coli K12 context. However, it did not encode a site-specific recombinase as mobile genomic islands usually do. It was then deduced that the host cells should provide the recombination function. This latter was searched for by means of a PCR approach to detect the island excision in E. coli K12 mutants affected in a number of recombination functions, including the 16 E. coli K12 site-specific recombinases, the RecET system, and multiple proteins that participate in the RecA-dependent pathways of homologous recombination. None of these appeared to be involved in the island excision. The second model, analyzed in a RecA deficient context, was a plasmid construction containing a short direct repeat proceeding from Saccharomyces cerevisiae, which flanked the cat gene. The excision of this gene by recombination of the DNA repeats was confirmed by PCR and through the detection, recovery and characterization of the plasmid deleted form. In sum, we present new evidence on the occurrence of RecA-independent recombination events in E. coli K12. Although the mechanism underlying these processes is still unknown, their existence suggests that RecA-independent recombination may confer mobility to other genetic elements, thus contributing to genome plasticity.

ReScribe: An Unrestrained Tool Combining Multiplex Recombineering and Minimal-PAM ScCas9 for Genome Recoding Pseudomonas putida

“…In the first place, the efficiency of recombineering varies with the relative location of the gene. In this context, cold and hotspots for recombineering have been identified in other studies . Additionally, the position of the target loci with respect to the two replichores may have an effect too.…”

“…In this context, cold and hotspots for recombineering have been identified in other studies. 64 Additionally, the position of the target loci with respect to the two replichores may have an effect too. Thus, genes closer to the origin of replication will be edited at higher levels than those located farther away.…”

“…Furthermore, ReScribe still has some of the limitations of the CRISPR technology, including (i) the need for constructing CRISPR plasmids directed at each modification locus; (ii) variations in the efficiency of the spacer, probably caused by differences in the secondary structure of the crRNA, which depends on the nucleotide composition of the spacer; 63 and (iii) the loss of functionality of the CRISPR plasmid caused by homologous recombination between the direct repeats or by mutating one of the CRISPR elements. 28,29,64 Regarding possible substitutions in the CRISPR-Cas9 counterpart, the recently engineered Sc 2+ and HiFi-Sc 2+ optimized ScCas9 variants could enhance the performance and robustness of ReScribe. 65 Lastly, the significant number of 40 off-target mutations was found in the genome sequence of KT2440Rc12.…”