Description: Background of our Project This year, we are focusing on the entomopathogenic fungus Metarhizium anisopliae, which is currently used as a biological control agent for different arthropods. However, its uses has been limited by poor efficacy. That is why many experts genetically engineered various M. anisopliae strains in order to improve its virulence. While genetic engineering is a great way to improve upon what mother nature has given us, biosafety has always been a big problem with genetic engineered organisms. For this reason, we hope to build a biosafety system, which includes one optogenetic module and one kill switch, to limit the persistence of our genetically engineered fungus in the environment. Details of our project In our biosafety system, our design consists of an optogenetic module and a kill switch. We used the optogenetic system VP-EL222 which containing the VP-EL222 genes and a promoter that activated by the VP-EL222 proteins blue light inducible dimerization and DNA binding. We only want the kill switch to be induced when the fungi have already killed the insect. To achieve this, we put VP-EL222 under the control of the hemolymph-induced promoter, Pmcl1. This allows the production of VP-EL222 proteins in the darkness of the insects’ interior. The fungus will breach the the dead host’s cuticle from inside after killing its host and expose to blue light, then lethality can be induced by built-in killswitch, which will be activated when the light-inducible promoter drives the expression of the proteins in the kill switch system due to VP-EL222 dimerization and the DNA binding. When it comes to the kill switch, we employ a simple, versatile, and filamentous-fungi-specific CRISPR/Cas9 system developed by the authors of the 2015 paper “A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi.” Because virtually any gene can be targeted by RNA-guided Streptococcus pyogenes Cas9, we target several genes that could disrupt the life cycle and reduce the survivability of the genetic engineered M. anisopliae. Two of our target genes, MrPHR1 and MrPHR2, encodes photolyases in Metarhizium. Removing them will seize the production of UV-induced cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) photoproducts [(6-4) PPs]. The effect is supported by 2012 paper “Enhanced UV Resistance and Improved Killing of Malaria Mosquitoes by Photolyase Transgenic Entomopathogenic Fungi” which shows that deleting native photolyase genes will strictly contain M. robertsii to areas protected from sunlight, alleviating safety concerns that transgenic hypervirulent Metarhizium spp. (We will blast the same genes in the M.anisopliae strain’s genome.) What NYMU team has been working on so far? We are still waiting for our Metarhizium anisopliae ARSEF549 which we booked from ARSEF, US. Thus, we conduct our experiences in E.coli. now. We have already completed the design of our system. However, we can't sure whether will our Metarhizium arrives on time or not. All we can do is to prepare all the other thing and as soon as the Metarhuzium arrived we can really start our project! What we hope to accomplish We have also chosen some other essential genes for M.anisopliae as our potential target genes. The repetible DNA regions in these essential genes will serve as the sgRNA template allowing the Cas9 to mutate many positions within the gene with high off-target activity, differing from mostly other CRISPR researches insisting Cas9 must have high on-target activity, resulting in the transformed M.anisopliae that will easily die under blue light.In conclusion, we want our project display one solution to the biosafety problem of genetically engineered entomopathogenic fungi. We also want to show the world that the CRISPR-Cas9 system is not just one of those lab tools that have no real impact on the lives of the non-scientific community, but a marvelous gene editing system that could change the daily lives of many people and help our strive towards a cleaner and better future.
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Year: 2016Visit Wiki
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Updated at: 8/9/16