Modification of the genetic backround of Mucor circinelloides using a plasmid free CRISPR/Cas9 system
The CRISPR/Cas9 genome editing system has been developed and optimized for several different organisms. In fungi, the transformation strategy includes a plasmid containing the crRNA, the trans-activating crRNA (tracrRNA) and the CRISPR-associated (Cas) nuclease, which are expressed together. In this study, we developed a plasmid free CRISPR/Cas9 system to modify the genetic background of Mucor circinelloides.
M. circinelloides is a carotene producing Mucoromycotina fungus used as a model organism in various molecular microbiological studies. Genetic manipulation of Mucoromycotina species based on homologous recombination is difficult to achieve and the mitotic stability of the resulting transformants is often low.
To optimize the plasmid free CRISPR/Cas9 system, we disrupted the M. circinelloides carB gene encoding the carotenogenic enzyme, phytoene dehydrogenase. PEG mediated protoplast transformation was used to introduce the Cas9 enzyme and a synthesized, carB specific gRNA with or without a DNA fragment containing the deletion cassette into the fungus. Successful disruption of the carB gene resulted in white colonies. If only the gRNA and Cas9 were transferred into the protoplasts, molecular analysis of the transformants indicated a 2300 nt gap upstream from the PAM sequence. If we co-transformed Cas9 and gRNA with the deletion cassette, the double-strand breaks of the DNA were repaired by homologous recombination.
After the optimization of the system, we have started to disrupt the three HMG-CoA reductase genes (hmgR1, hmgR2 and hmgR3) of M. circinelloides separately and together. HMG-CoA reductase catalyses the rate-limiting step of the isoprene biosynthesis. Our results suggested that hmgR1 plays an important role in the adaptation to the low temperature while disruption of hmgR2 and hmgR3 resulted in increased sensitivity to statins. The latter genes proved to be determining for the general isoprenoid biosynthesis as their combined disruption was lethal.
The study was supported by the grants LP2016-8/2016 and GINOP-2.3.2-15-2016-00035.