A Simple and Efficient CRISPR Technique for Protein Tagging

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Homology-directed repair (HDR) for genetic knock-in is often inefficient.
• Current methods require selection of modified cells, limiting primary cell applications.

Purpose of the Study:

• To develop a novel, highly efficient method for CRISPR-mediated protein tagging.
• To overcome the limitations of HDR in genetic modification of cells.

Main Methods:

• Introduction of Homology independent gene Tagging (HiTag) system.
• CRISPR-Cas9 mediated gene tagging in various cell types.
• Electroporation for efficient delivery of reagents.

Main Results:

• HiTag achieved protein tagging in up to 66% of transfected cells.
• Demonstrated effectiveness across diverse cell types, including primary cells.
• Successfully applied HiTag for fluorescent protein knock-in, protein localization studies, and luciferase assays.

Conclusions:

• HiTag provides a robust and efficient alternative to HDR for genetic knock-in.
• The method facilitates live cell imaging, protein localization, and protein level monitoring.
• HiTag expands the utility of CRISPR technology in primary cells and various research applications.