Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• The prokaryotic CRISPR/Cas9 system is a powerful tool for genome editing.
• CRISPR/Cas9 has been adapted for use in various cells and model organisms.

Purpose of the Study:

• To describe targeted gene deletion and replacement in human cells using the CRISPR/Cas9 system with two guide RNAs.
• To evaluate the efficiency and precision of CRISPR/Cas9-mediated genome editing.

Main Methods:

• Utilized the CRISPR/Cas9 system with two guide RNAs for targeted gene manipulation in human cells.
• Employed a homology repair donor template for precise gene replacement experiments.

Main Results:

• Achieved efficient targeted gene deletions of various lengths, irrespective of the target gene's transcriptional status.
• Demonstrated high efficiency in forming correct junctions for targeted gene deletions.
• Successfully guided precise gene replacement when a homology repair donor was provided.

Conclusions:

• The CRISPR/Cas9 system effectively and precisely generates targeted gene deletions and replacements in human cells.
• This methodology facilitates the characterization of functional domains in protein-coding genes and noncoding regulatory sequences.
• CRISPR/Cas9 is a valuable tool for advancing genomic research in human and animal systems.