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CRISPR-Cas9 gene editing in honeybee and pig

Research output: Book/anthology/dissertation/reportPh.D. thesis


  • Anja Pen
Creating animal models by using genome modification has gotten significantly more accessible thanks to the CRISPR-Cas9 technique. In this study, we aimed to the implement the CRISPR-Cas9 methodology in the European honeybee (Apis mellifera) and pig (Sus scrofa) for generation of animal models. We want to use these animal models to study the development of honeybees and the pathology of amyotrophic lateral sclerosis (ALS) in a pig model of human disease. In order to simplify the production of these animal models, we test the use of sperm mediated gene transfer (SMGT) in combination with CRISPR-Cas9.
Honeybees are highly eusocial insects that develop specific phenotypical features because of differential feeding regimes in the larval stage. Since the genetic basis of each honeybee is similar, these features result from a change in the epigenome of the insects. Investigating the underlying mechanisms of honeybee development using genome modification will aid in uncovering these complex genetic regulatory systems. In honeybees, we have attempted to induce genome modification in the cinnabar gene through microinjection and feeding of CRISPR-Cas9 components to larvae. Additionally, we tested whether Cas9 is able to alter the genome of honeybee spermatozoa.
Pigs have been used as models for human disease for many years but creating these models has been challenging. Our research group is mainly interested in modelling neurodegenerative diseases, such as Parkinson´s disease and ALS. We aim to produce model animals using the CRISPR-Cas9 method in combination with SMGT. In pig, the goal was to produce a genetically modified animal model of SOD1 with CRISPR-Cas9 components transferred through SMGT. To this end, we have tested Cas9 editing in porcine cell lines and inseminated sows with semen containing a Cas9-sgRNA DNA construct. In our animal models, we would like to study the RNA binding properties of proteins because of its involvement RNA handling in the pathology of ALS. Therefore, we also tested a method called RNP immunoprecipitation and sequencing (RIP-seq).
We have not succeeded in generating genome modifications at our target sites, which means that the implementation of the methods is not yet completed. However, we have gained valuable knowledge about the CRISPR-Cas9 system and RIP-seq that we can build future experiments on.
Original languageEnglish
Number of pages126
Publication statusPublished - 31 May 2018

Note re. dissertation

Ph.d.-grad tildelt: 24-09-2018

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