CRISPR-Cas9 Editing of the Synthesis of Biodegradable Polyesters Polyhydroxyalkanaotes (PHA) in Pseudomonas putida KT2440.

Genome editing technologies allow us to study the metabolic pathways of cells and the contribution of each associated enzyme to various processes, including polyhydroxyalkanoate (PHA) synthesis. These biodegradable polyesters accumulated by a range of bacteria are thermoplastic, elastomeric, and biodegradable, thus have great applicative potential. However, several challenges are associated with PHA production, mainly the cost and shortcomings in their physical properties. The advances in synthetic biology and metabolic engineering provide us with a tool to improve the production process and allow the synthesis of tailor-made PHAs. CRISPR/Cas9 technology represents a new generation of genome editing tools capable of application in nearly all organisms. However, off-target activity is a crucial issue for CRISPR/Cas9 technology, as it can cause genomic instability and disruption of functions of otherwise normal genes. Here, we provide a detailed protocol for scarless deletion of the genes implicated in PHA metabolism of Pseudomonas putida KT2440 using modified CRISPR/Cas9 systems and methodology.