User-Friendly Genetic Conditional Knockout Strategies by CRISPR/Cas9

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• Loss-of-function studies are crucial for understanding gene function in model organisms and cells.
• Conditional gene inactivation in diploid cells is challenging due to complex procedures like vector construction and electroporation.

Purpose of the Study:

• To present a simplified strategy for generating biallelic conditional gene and DNA regulatory region knockouts.
• To facilitate functional studies of essential genes and regulatory regions during development.

Main Methods:

• Utilized CRISPR-Cas9 technology combined with short-homology-arm targeting vectors.
• Employed sequential or simultaneous codelivery of CRISPR-Cas9 and targeting vectors in mouse embryonic stem cells.
• Developed a rapid method for conditional knockout of DNA elements.

Main Results:

• Successfully generated biallelic conditional knockouts of genes and regulatory regions.
• Demonstrated a simplified and rapid approach compared to traditional methods.
• The method is applicable to any DNA element requiring conditional inactivation.

Conclusions:

• The presented strategy offers a simple and rapid method for conditional gene and DNA regulatory region knockouts.
• This approach significantly facilitates the functional analysis of essential genes and regulatory regions in developmental studies.