Many of the most conserved sequences in the human and mouse genomes overlap with poison exons, which disrupt the open reading frames of protein-coding genes. The team of Dr. Robert Bradley at Fred Hutchinson Cancer Research Center has shown that many of these poison exons are required for the growth of cultured cells. However, scientists don’t yet know whether poison exons are required for organismal viability.
To answer this question, Bradley’s team needed to create a mouse model of poison exon loss. However, such a model had never been created before, and doing so would be a challenge. That’s because it would require, first, the precise deletion of a splice site to prevent the poison exon’s inclusion without the disruption of other potentially important genomic elements. Secondly, it would be necessary to generate related control models, such as a model with the deletion of a similarly sized element within the flanking intron.
Bradley worked with the Preclinical Modeling core’s genetically engineered mouse model (GEMM) team, led by staff scientist Dr. Priti Singh, to create the technically challenging models he needed. Drawing on her years of experience generating CRISPR-edited mouse models, Singh designed an experimental strategy and implemented it in collaboration with GEMM microinjectionist Areum Yoon.
The GEMM team successfully created three Smndc1 poison exon knockout lines in just two and a half months — much faster than Bradley had anticipated based on his previous work with private mouse-modeling companies. Specifically, they used a paired-guide strategy to remove the entire exon in question by microinjecting gRNA and the Cas9 complex into the male pronucleus of one-cell mouse embryos. They then transferred the gene-edited embryos into surrogate mothers.
The project resulted in what the investigators believe is the first-ever mouse model of the deletion of an ultraconserved poison exon, plus several other related models. These models have enabled the Bradley Lab to ask entirely new biological questions in physiological settings.
Read more about the Bradley Lab’s research.