Genome Editing As an Approach to the Study of in Vivo Transcription Reprogramming

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• CDK8 kinase activity is crucial for gene expression and transcriptional reprogramming.
• Complete knockout of the cdk8 gene is lethal in early embryonic development (morula stage).
• Modeling CDK8's role in adult organisms requires methods to attenuate its activity without causing lethality.

Purpose of the Study:

• To develop a mouse model with attenuated CDK8 kinase activity for studying transcriptional reprogramming in adult organisms.
• To introduce a specific F97G mutation in the cdk8 gene's catalytic domain.
• To assess the efficacy of CRISPR/Cas9 gene editing for site-specific mutation introduction.

Main Methods:

• Utilized CRISPR/Cas9 technology for precise genome editing in mice.
• Introduced a double-stranded DNA break 128 base pairs upstream of the target mutation site.
• Employed a homologous repair template with specific arms to insert the F97G mutation into exon 3 of the cdk8 gene.

Main Results:

• Successfully generated viable mice carrying the site-specific F97G mutation in the cdk8 gene.
• Demonstrated high editing efficiency for mutations located 128 base pairs from the double-strand break site.
• Obtained the first live animals with attenuated CDK8 kinase activity via the F97G mutation.

Conclusions:

• The F97G mutation effectively attenuates CDK8 kinase activity, enabling the study of its role in adult transcriptional reprogramming.
• This novel mouse model provides a valuable tool for investigating gene expression regulation and related processes.
• The study highlights the effectiveness of CRISPR/Cas9 for precise gene editing at a distance from the induced break.