CRISPR/Cas9 Genome Editing by Electrotransformation in Denitrificimonas caeni sp.
Abstract
Changes in climate are an increasing threat to society, with carbon dioxide (CO2), methane, and nitrous oxide (N2O) being seen as the most significant contributing greenhouse gases. N2O has a global warming potential 300 times that of CO2. Agriculture is the largest anthropogenic source of N2O, with 65% of emissions due to the application of nitrogen fertilizers. Proposed reduction measures have mainly focused on nitrogen-use efficiency (NUE), but recent research suggests that influencing the microbiome in agricultural fields may have a higher reduction potential. Microorganisms in soil can, in the absence of oxygen, use nitrogen compounds to continue their respiration. This denitrification process is a stepwise reduction of NO3- ® NO2- ® NO ® N2O ® N2, respectively catalyzed by the enzymes NAR/NAP, NIR, NOR, and NOS. Some organisms can perform a complete reduction from NO3- to N2, while others can only perform partial reduction. Organisms that can only perform partial reduction lack functioning enzymes for some of these steps. Organisms that only have NOS, Non-denitrifying N2O-respiring bacteria (NNRB), will be net N2O consumers, and an increase in the number of NNRB in fields could reduce N2O emissions during fertilization.
Prior to this thesis, selection for survival in soil and organic waste (the dual substrate enrichment method) was used to find suitable NNRB. The work resulted in 20 isolates with NOS, but several also had NIR and NOR. This means that they can both produce and reduce N2O and therefore have a weaker ability to reduce nitrous oxide emissions. But by knockout of the NIR gene in these organisms, they would become strong candidates for reducing nitrous oxide emissions from soil. In this thesis, I have tried to perform such a knockout using CRISPR/Cas9. The experiments were performed on the isolate Denitrificimonas caeni sp. TC7.
The CRISPR/Cas9 system used in this experiment was a two-plasmid system where pCasPA expresses Cas9 nuclease and pACRISPRΔnirS expresses sgRNA and contains the template for homology directed repair. A broad host range vector pL2020 would be used to reintroduce NIR in the knockout mutant to show that the ability to reduce NO2- to NO is due to this enzyme. To transform the plasmids in the organism I used electroporation. As this was not possible despite several attempts under different conditions, I chose to test electroporation transformation with pL2020 and quantify the efficiency at different electroporation intensities. I worked quantitatively, and quantified survival (plate spread on non-selective medium) and the number of viable transformants (plate spread on medium with chloramphenicol). The number of transformants reached a maximum at an intensity that killed about 50 % of the cells. By increasing the voltage to 1800 V mm-1, only 5-6 % of the cells survived, and the total number of viable transformants dropped drastically. However, the number of transformants per surviving cell was higher at 1800 than at 1500 V mm-1. The maximum transformation efficiency (at 1500 V mm-1) was low: about six transformants per 109 viable cells. The molecular efficiency, i.e. the number of transformants per number of plasmids, was also low: about 2·10-10 transformants per plasmid. This is extremely low compared to other published results, suggesting that Denitrificimonas caeni sp. TC7 is difficult to transform by electroporation with pL2020 and other approaches should be tested. Additionally, cell death and transformation kinetics was simulated with a simple model which was parameterized by least square (modelled versus measured survival and transformation frequency). This type of modelling could be useful for further electroporation attempts.