Implementation and Gene Editing of the Microorganism Rhodopseudomonas Palustris Trough the CRISPR Cas9 System

Authors: 
García-Peña, E. I., Instituto Politécnico Nacional
Salgado Manjarrez, E., Unidad Profesional Interdisciplinaria de Biotecnología, Instituto Politecnico Nacional
Jurado Marbán, V. H., Unidad Profesional Interdisciplinaria de Biotecnología, Instituto Politecnico Nacional
This work shows the application and implementation of the CRISPR Cas9 method to edit the genome of a microorganism that lacks the validated PAM and tracrRNA sequences required for the CRIPSR Cas9 gene editing tool (Ma et al, 2015). The purple non-sulfur bacteria used in this work is the highly adaptable Rhodopseudomonas Palustris, a microorganism capable of shifting between the four modes of metabolism that support life. This on itself, gives the microorganism a remarkable potential given the large amount of high value metabolites it can generate. It can produce hydrogen (H2), polyhydroxyalkanoates (PHA) and pigments, which are promising given their vast amount of applications and value (Laimer et al,2004). However as mentioned by other authors, the extraordinary adaptability the microorganism presents can become a major drawback, as it becomes exceedingly difficult to redirect the metabolism in a highly specific way (Kim et al, 2012; Natha et al, 2009; Wu et al, 2012; Lee et al, 2002). for instance, R. Palustris relies on the reducing power available to produce several metabolites and the metabolic routes for these often compete among them, the distribution of the reducing power promotes a competition between the production of PHB and hydrogen, or growth (Kim et al, 2012; McKinlay et al, 2013). Considering the previously stated, genetic manipulation of the microorganism is interesting since it is possible to maximize the production of a specific metabolite while reducing or eliminating the undesired. In order to perform an efficient and intelligent modification of the microorganism, the main metabolic routes involved in the production of the metabolites must be closely analyzed, in accordance to this point several authors report the performance of mutant strains with one or several modifications to said metabolic pathways(H2, PHB and pigments) (Chen et al, 2012; Heiniger et al, 2011; Laguna et al, 2011) this information will facilitate the selection of the genetic modifications; also by realizing a gene expression analysis on R. Palustris during different culture conditions is possible to increase the efficacy of the overall process. To edit the genome of the microorganism the type II CRISPR cas9 system was selected due to the minimalist architecture of the mechanism and the elevated likelihood of Rhodopseudomonas having the type II-C CRISPR cas9 system (Burstein et al 2017).