Highly Efficient Genome Editing via CRISPR/Cas9 to Create Clock Gene Knockout Cells

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Chronobiology

Background:

• CRISPR/Cas9 is a powerful genome editing tool for functional studies.
• Circadian rhythm research has not extensively utilized CRISPR/Cas9.
• FBXL3 is implicated in regulating circadian clock function.

Purpose of the Study:

• To apply CRISPR/Cas9 for generating an Fbxl3 knockout in a human cell line.
• To investigate the effects of Fbxl3 knockout on circadian rhythms.
• To assess the utility of CRISPR/Cas9 for circadian biology research.

Main Methods:

• CRISPR/Cas9 genome editing was used to target the Fbxl3 gene.
• High efficiency and specificity of genomic alterations at the Fbxl3 locus were achieved.
• Analysis of Bmal1-luciferase reporter activity and CRY1 protein stability in knockout cells.

Main Results:

• CRISPR/Cas9 mediated Fbxl3 knockout with 70%-100% efficiency.
• Fbxl3 knockout cells exhibited low amplitude and long period oscillations of Bmal1-luciferase.
• Increased CRY1 protein stability was observed in Fbxl3 knockout cells.

Conclusions:

• CRISPR/Cas9 is highly effective for generating gene knockouts in human cells.
• Fbxl3 knockout significantly impacts circadian rhythm oscillations and protein stability.
• CRISPR/Cas9 is a valuable tool for studying circadian rhythms across various model systems.