3rd International Conference on CRISPR Technologies
CRISPR/Cas9 meets recombineering in P. putida KT2440
Authors
Tomas Aparicio - Presenter, Centro Nacional de Biotecnologඡ (CNB-CSIC)
Vactor de Lorenzo, Centro Nacional de Biotecnologඡ (CNB-CSIC)
Esteban Martanez-Garcaa, Centro Nacional de Biotecnologඡ (CNB-CSIC)
The implementation of single-stranded DNA recombineering technology in Pseudomonas putida KT2440 has been proven as a rapid and reliable method for genome editing of this platform strain(1). A drawback of this methodology is the low frequency at which the desired changes are introduced in the genome, making it difficult to isolate mutants displaying a non-selectable phenotype. The adoption of CRISPR/Cas9 is an efficient way to counterselect wild-type cells that have not undergone the genetic modifications introduced by ssDNA recombinering, so the combination of both methods dramatically improves the output of this technology, boosting the genome editing capabilities available for P. putida KT2440. In this work we report the incorporation of the functional modules for recombineering (ssr recombinase) and CRISPR/Cas9 (Cas9, tracrRNA and the CRISPR array) into pSEVA plasmids to generate a platform that allows a suite of genomic changes ranging form single nucleotide substitutions to large genomic deletions, including the simultaneous deletion of two independent genomic locus(2). An upgrade of the system is also presented, featuring: i) the sevarization of the functional modules ii) a cycling method to sequentially perform deletions without curing the working plasmids. The latest attempts to improve this technology will be also presented, namely the condensation of CRISPR/Cas9/recombineering elements in a single plasmid.