Rapid Modeling of Developmental Disorders Using CRISPR/ Cas9
In: Student Reports, Summer 2018, The Jackson Laboratory
Dr. Kevin Peterson and Dr. Steve Murray
CRISPR/Cas9 technology has greatly enlianced the scope and pace of genome editing due to its ease of implementation and programability. The generation of precision alleles, such as introducing point mutations or small insertions, relies on the incorporation of donor template DNA by homology directed repair (1-IDR). Despite significant advancements in the field, HDR efficiency remains low in zygotes. In my project, I sought to optimize mutagenesis efficiency and the HDR pathway by investigating the function of different Cas9 ribonucleoprotein complexes and coordinating the timing of Cas9 activity with the cell cycle. In addition, I worked on the development and implementation of a fluorescence-based assay to quickly evaluate success of HOR. In an FO proof-of-principle study, I applied the knowledge gained from the optimization experiments to model a novel human mutation in TPMl associated with congenital heart defects. When testing electroporation parameters, we found that increasing pulse duration from 1 ms to 3 ms had a greater impact on mutagenesis efficiency compared to cl1anging pulsenumber from six to twelve. Additionally, we observed increased editing with sgRNA Cas9 RNP complex compared to Alt-R guides Cao;9 RNP complex. In our preliminary implementation of GFP-BFP conversion assay, we utilized the R26-m'fmG reporter strain and successfully excised the mtdTomato reporter using TAT-Cre revealing the membrane bound GFP. In summary, this work will contribute to increasing both on-target cutting efficiency and HDR through zygote electroporation, which will greatly enhance the analysis of developmental phenotypes in founder (FO) embryos.
Ladha, Ali, "Rapid Modeling of Developmental Disorders Using CRISPR/ Cas9" (2018). Summer and Academic Year Student Reports. 2609.