High-efficiency multi-site genomic editing (HEMSE) ofPseudomonas putidathrough thermoinducible ssDNA recombineering

Abstract

SUMMARYWhile single-stranded DNA recombineering is a powerful strategy for higher-scale genome editing, its application to species other than enterobacteria is typically limited by the efficiency of the recombinase and the action of native mismatch repair (MMR) systems. By building on [i] availability of the Erf-like single-stranded DNA-annealing protein Rec2, [ii] adoption of tightly-regulated thermoinducible device and [iii] transient expression of a MMR-supressing mutL allele, we have set up a coherent genetic platform for entering multiple changes in the chromosome of Pseudomononas putida with an unprecedented efficacy and reliability. The key genetic construct to this end is a broad host range plasmid encoding co-transcription of rec2 and P. putida’s mutLE36KPP at high levels under the control of the PL/cI857 system. Cycles of short thermal shifts of P. putida cells followed by transformation with a suite of mutagenic oligos delivered different types of high-fidelity genomic changes at frequencies up to 10% per single change—that can be handled without selection. The same approach was instrumental to super-diversify short chromosomal portions for creating libraries of functional genomic segments—as shown in this case with ribosomal binding sites. These results enable the multiplexing of genome engineering of P. putida, as required for metabolic engineering of this important biotechnological chassis.

Improved bacterial recombineering by parallelized protein discovery

“…Two of these recombineering-based methods, multiplex automated genome engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), have been used for a variety of high-value applications (3,(10)(11)(12)(13)(14), but at their core they offer the ability to generate populations of bacteria that can contain billions of precisely targeted mutations. However, while these techniques function well in E. coli and some closely related enterobacteria, efforts to reproduce these results in other bacterial species have been sporadic and stymied by low efficiencies (SI Appendix, Table S1) (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31).…”

CRISPR-Assisted Multiplex Base Editing System in Pseudomonas putida KT2440

“…However, traditional genetic tools in P. putida KT2440 are difficult to manipulate and inefficient. The emergence of the CRISPR/Cas9 genome editing system has greatly simplified genetic engineering of P. putida KT2440, but still requires the provision of heterologous repair proteins and donor DNA templates (Aparicio et al, 2018;Sun et al, 2018). Additionally, simultaneous genome editing of two loci remains extremely challenging using the CRISPR/Cas9 system.…”

“…A unique characteristic of the CRISPR/Cas9 system is the feasibility of concurrent multiplex genome editing, yet genome editing of two loci or more in eukaryotes has been the primary focus (Jakoèiunas et al, 2016) and few studies of multiplex genome editing have been reported for prokaryotes, which can be ascribed to the weak HDR and poor or lack of non-homologous end joining (NHEJ) repair (Wu et al, 2019b). Although multiplex genome editing of two loci by the CRISPR/Cas9 system had been tested in P. putida KT2440, the efficiency was extremely low and could not be applied readily in experiments (Aparicio et al, 2018).…”

“…In recent years, Cas9 assisted genome editing systems have revolutionarily accelerated the development of genetic studies in different organisms (Jakoèiunas et al, 2016), including Pseudomonas spp. (Aparicio et al, 2018;Sun et al, 2018;Wu et al, 2019a). Nevertheless, the developed P. putida CRISPR/Cas9 systems require the introduction of a heterologous recombination system because of inefficient homology-directed repair (HDR), and the provision of donor DNA templates or single-stranded DNA is also not straightforward (Aparicio et al, 2018;Sun et al, 2018).…”

“…(Aparicio et al, 2018;Sun et al, 2018;Wu et al, 2019a). Nevertheless, the developed P. putida CRISPR/Cas9 systems require the introduction of a heterologous recombination system because of inefficient homology-directed repair (HDR), and the provision of donor DNA templates or single-stranded DNA is also not straightforward (Aparicio et al, 2018;Sun et al, 2018). Additionally, the superior characteristics of the CRISPR/Cas9 system in multiplex genome editing has only been achieved in two loci with extremely low efficiency (Aparicio et al, 2018).…”