CRISPR-Assisted Multiplex Base Editing System in Pseudomonas putida KT2440

Tian-Xin Liang,Li-Bing Lu,Li-Rong Yang,Jun Sun,Jian-Ping Wu

doi:10.3389/fbioe.2020.00905

Tian-Xin Liang, Li-Bing Lu

Open Access

https://doi.org/10.3389/fbioe.2020.00905

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Abstract

Pseudomonas putida (P. putida) KT2440 is a paradigmatic environmental-bacterium that possesses significant potential in synthetic biology, metabolic engineering and biodegradation applications. However, most genome editing methods of P. putida KT2440 depend on heterologous repair proteins and the provision of donor DNA templates, which is laborious and inefficient. In this report, an efficient cytosine base editing system was established by using cytidine deaminase (APOBEC1), enhanced specificity Cas9 nickase (eSpCas9ppD10A) and the uracil DNA glycosylase inhibitor (UGI). This constructed base editor converts C-G into T-A in the absence of DNA strands breaks and donor DNA templates. By introducing a premature stop codon in target spacers, we successfully applied this system for gene inactivation with an efficiency of 25–100% in various Pseudomonas species, including P. putida KT2440, P. aeruginosa PAO1, P. fluorescens Pf-5 and P. entomophila L48. We engineered an eSpCas9ppD10A-NG variant with a NG protospacer adjacent motif to expand base editing candidate sites. By modifying the APOBEC1 domain, we successfully narrowed the editable window to increase gene inactivation efficiency in cytidine-rich spacers. Additionally, multiplex base editing in double and triple loci was achieved with mutation efficiencies of 90–100% and 25–35%, respectively. Taken together, the establishment of a fast, convenient and universal base editing system will accelerate the pace of future research undertaken with P. putida KT2440 and other Pseudomonas species.

Highlights

Pseudomonas spp. are well-known gram-negative environmental bacteria, which contain more than 200 species (Nikel et al, 2014)
Using the plasmid pSEVA-gRNAF (Sun et al, 2018) as the template, a N20 sequence (TtgA spacer targeting the ttgA gene in KT2440) was used as the target site and inserted into pSEVA-gRNAF to give pSEVA-TtgA
Pseudomonas putida KT2440 is a potential chassis for industrial production of bio-based materials, pharmaceuticals and chemicals (Nikel et al, 2016)

Summary

Introduction

Pseudomonas spp. are well-known gram-negative environmental bacteria, which contain more than 200 species (Nikel et al, 2014). The research area of Pseudomonas spp. can be divided mainly into three fields: Non-pathogenic Pseudomonas putida (P. putida) KT2440 is used as a chassis for synthetic biology, metabolic engineering and biocatalysis (Poblete-Castro et al, 2012); Multiplex Base Editing System the opportunistic pathogen P. aeruginosa is regarded as a model strain for investigation of antibiotics resistance and disinfectants (Stover et al, 2000); the plant commensal P. fluorescens is well known for its biological control properties (Paulsen et al, 2005). The developed P. putida CRISPR/Cas 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 not straightforward (Aparicio et al, 2018; Sun et al, 2018). The superior characteristics of the CRISPR/Cas system in multiplex genome editing has only been achieved in two loci with extremely low efficiency (Aparicio et al, 2018)

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Conclusion