High-throughput screens in mammalian cells using the CRISPR-Cas9 system

Area of Science:

• Genomics
• Molecular Biology
• Genetic Engineering

Background:

• The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is a revolutionary genome-editing technology.
• Its application has rapidly expanded into large-scale functional screening in mammalian cells.

Purpose of the Study:

• To review the CRISPR-Cas9 library-mediated functional screening procedure.
• To discuss system optimization strategies and applications of this genetic toolkit.

Main Methods:

• Construction of genetic libraries using lentiviral delivery of single-guide RNA collections.
• Directing Cas9 or dead Cas9-effector fusions to interrogate gene function or regulate transcription.
• Application in both loss-of-function and gain-of-function screening.

Main Results:

• CRISPR-Cas9 screening demonstrates superior effectiveness and reliability compared to RNA interference.
• CRISPR-Cas9 libraries can target protein-coding sequences and regulatory elements (promoters, enhancers, ncRNA elements).

Conclusions:

• The CRISPR-Cas9 system accelerates mechanistic discovery in biological processes.
• This genetic toolkit provides a powerful platform for comprehensive functional genomics studies.