Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system

Abstract

Background: The soil bacterium Pseudomonas putida KT2440 is a "generally recognized as safe"-certified strain with robust property and versatile metabolism. Thus, it is an ideal candidate for synthetic biology, biodegradation, and other biotechnology applications. The known genome editing approaches of Pseudomonas are suboptimal; thus, it is necessary to develop a high efficiency genome editing tool.

Results: In this study, we established a fast and convenient CRISPR-Cas9 method in P. putida KT2440. Gene deletion, gene insertion and gene replacement could be achieved within 5 days, and the mutation efficiency reached > 70%. Single nucleotide replacement could be realized, overcoming the limitations of protospacer adjacent motif sequences. We also applied nuclease-deficient Cas9 binding at three locations upstream of enhanced green fluorescent protein (eGFP) for transcriptional inhibition, and the expression intensity of eGFP reduced to 28.5, 29.4, and 72.1% of the control level, respectively. Furthermore, based on this CRISPR-Cas9 system, we also constructed a CRISPR-Cpf1 system, which we validated for genome editing in P. putida KT2440.

Conclusions: In this research, we established CRISPR based genome editing and regulation control systems in P. putida KT2440. These fast and efficient approaches will greatly facilitate the application of P. putida KT2440.

Keywords: CRISPR–Cas9 system; CRISPR–Cpf1 system; Genome editing; Pseudomonas putida KT2440; Single nucleotide mutation; Transcriptional engineering.

Fig. 1

Strategy for the construction of a CRISPR–Cas9 two-plasmid system in P. putida KT2440. a pSC101 replicon in pCASsac was replaced with RK2 replicon together with oriT fragment, creating pCAS-RK2K. In pCAS-RK2K, cas9 gene was linked with its native promoter, and gRNA cassettes were transcribed by P_rhaB promoter so as to guide Cas9 protein targeting pRO1600 replicon in pSEVA-gRNAT. The λ-Red recombination system was under control of arabinose promoter to enhance the repairing efficiency in KT2440. SacB, a commonly used counterselectable marker function as a self-curing tool. b The P_trc-lacl^q inducible system was eliminated from pSEVA644 and gRNA cassettes were inserted, generating pSEVA-gRNAF. The upstream homologous arm (UHA) and downstream homologous arm (DHA) were amplified from genome and connected by overlap-extension PCR. Next, the combinational homologous arm was assembled into pSEVA-gRNAF, giving rise to pSEVA-gRNAT

Fig. 2

CRISPR–Cas9 mediated nicC gene deletion in the Pseudomonas putida KT2440. a The phenotypes of pSEVA-gRNAT derivatives transformed into KT2440 harboring pCAS-RK2K. All plasmids were electrotransformed into pCAS-RK2K cells with an equal amount of DNA. b The schematic represents the design of identification primers for nicC gene deletion. Yellow arrow means the location of N20 sequence in nicC gene. Blue arrow represents the location of identification primers NT-JF and NT-JR. c Agarose gel electrophoresis shows the result of colony PCR to confirm nicC gene editing efficiency. d DNA sequencing proves that the 1149-nt nicC gene have been successfully deleted

Fig. 3

Schematic diagram of the essential effects among four components (Cas9 protein, gRNA cassette, homologous arms and λ-Red system) in the Pseudomonas putida KT2440 genome editing. a The strategy of plasmids with different components were transformed into KT2440. pCAS-RK2K or its derivatives was first transformed into KT2440. In the second round of electroporation, pSEVA644, pSEVA-gRic6T and its derivatives were transformed into KT2440 harboring pCAS-RK2K relevant plasmids. b Electroporation efficiency is reflected from the total number of CFU (colony-forming units); Mutation efficiency in six groups with different components. The CFU experiment is obtained from three replicates. Cells were plated on the equal concentration antibiotics plates and the amount of DNA was equivalent in each experiment

Fig. 4

The two-step strategy of single nucleotide mutation for PAM unavailability sites using CRISPR–Cas9 system. a The N20 sequence from pSEVA-NicA20 was used as Cas9 cutting site in KT2440, and the Gln139 in nicC gene was targeted as mutation site by mutating CAA to CTA. Yellow star means the target nucleotide. b After the first step genome editing, an added artificial N20 sequence (A20 sequence) and single nucleotide mutation (At Gln139 by mutating CAA to CTA) in pSEVA-NicA20 homologous arm were inserted into KT2440 genome. c Through the first step, single nucleotide mutation was applied into target locus. Then, we attempted to eliminate A20 sequence by curing pSEVA-NicA20 and using pSEVA-NicA21 for next genome editing step. In pSEVA-NicA21, the target site was changed from N20 sequence to A20 sequence. In the homologous arm, A20 sequence was eliminated and single nucleotide mutation was retained

Fig. 5

CRISPR–dCas9 mediated transcription inhibition in the Pseudomonas putida KT2440. a Schematic representation of pCAS-ZE0 and its derivatives (pCAS-ZE1, pCAS-ZE2, pCAS-ZE3) used for transcription inhibition. Plasmid-borne enhanced green fluorescence protein (eGFP) was selected as target site. b Illustration of different dCas9 binding sites are indicated in the upstream sequence of plasmid pSEVA-eGFP. ZE1 and ZE2 were targeting the − 35 region of J5 promoter, and ZE3 was binding with Ribosome Binding Site (RBS). To examine the effect of selecting different DNA strand, ZE1 was designed to bind with template strand and ZE2 was located at the non-template strand. c KT2440 cells harboring dCas9 and eGFP plasmids were gathered with equal amount and exposed under UV light. Blank KT2440 cells were used as control. d Comparsion of the repression effectiveness of dCas9 binding with different target sites

Fig. 6

CRISPR–Cpf1 mediated genome editing in the Pseudomonas putida KT2440. a Overview of the genome editing by CRISPR–Cpf1 in P. putida KT2440. b The schematic showing the design of identification primers to confirm genome editing in KT2440. c Agarose gel electrophoresis and the result of DNA sequencing show that pCpf1-RK2K enables gene deletion in KT2440

Fig. 7

Diagram for the CRIPSR–Cas9-assisted genome editing in P. putida KT2440. Day 1: Introduce the pCAS-RK2K plasmid into P. putida KT2440, and then inoculate the transformants in LB medium overnight; Day 2: Transfer the cultivated cells into fresh LB medium and add arabinose to trigger expression of λ-Red proteins. Next, cells were prepared as elecompetent cells and the pSEVA-gRNAT plasmids were transferred to KT2440 harboring pCAS-RK2K. Day 3: Screen out the mutants by colony PCR. The mutants were inoculated in LB medium containing antibiotic and rhamnose in the morning; Streak the cultivated cells on LB agar in the evening; Day 4: Screen out the mutants that have lost the pSEVA-gRNAT plasmids, and inoculate the mutants in LB medium containing glucose and sucrose. Next, in the evening, the cultivated cells were streak on plate containing glucose and sucrose, and then cultivated overnight. The mutants that have been cured of pSEVA-gRNAT can be used for the next round of genome editing; Day 5: Identify the mutants from the selection plate