Document Type

Poster

Publication Date

4-30-2020

Institution/Department

Maine Medical Center, Medical Education, Maine Medical Center Research Institute

MeSH Headings

Mice, Animals, Clustered Regularly Interspaced Short Palindromic Repeats, Alleles, CRISPR-Cas Systems, Gene Editing

Abstract

There is high demand for the development of experimental mouse models to study physiological processes and human diseases. CRISPR/Cas technology has offered a relatively efficient way to generate modifications in the mouse genome. However, this methodology often creates multiple, diverse genetic alterations, even when a strategy for a very specific targeted mutation is implemented. It is also rare that F0 mice will have single modified allele, and commonly, resultant mice are mosaic, containing multiple genomic modifications. In order to reduce the time and resources required to screen F0 mice and to detect somatic mosaicism, we implemented use of the computational analysis tool ICE. ICE is a CRISPR editing tool developed to analyze genomic modification in pooled cultured cells, where multiple genomic modifications can be expected. We applied this tool to determine its value in predicting genomic modifications in vivo using CRISPR/Cas. We tested one experimental case where sgRNA guided Cas9 created a double stranded DNA break in the Dio3 gene, and a ssDNA oligo provided as a template to insert a genetic tag. Preliminary data indicate a good correlation between genomic modifications detected by conventional Sanger sequencing of clones and those predicted by ICE. By increasing the number of direct comparisons between ICE predictions and actual sequence results, we aim to develop a reliable protocol to detect and evaluate efficiency of genomic modifications in F0 mice with assistance of computational methods.

Comments

2020 Costas T. Lambrew Research Retreat

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