Introducing Large Genomic Deletions in Human Pluripotent Stem Cells Using CRISPR-Cas3

Area of Science:

• * Molecular Biology
• * Gene Editing
• * Genomics

Background:

• * CRISPR-Cas systems are essential tools for eukaryotic genome editing and therapeutic applications.
• * Current CRISPR technologies (Type II/V) are limited in creating large targeted genomic deletions.
• * Type I CRISPR systems, utilizing Cascade and Cas3, offer unique enzymatic features for extensive DNA manipulation.

Purpose of the Study:

• * To present detailed protocols for generating large genomic deletions using the Type I CRISPR-Cas3 system in human cells.
• * To enable precise removal of DNA segments for therapeutic and research applications.
• * To provide a method for interrogating structural variants and noncoding elements impacting gene function and disease.

Main Methods:

• * Purification of Thermobifida fusca Cascade and Cas3 proteins.
• * Introduction of purified CRISPR-Cas3 ribonucleoprotein complexes into human cells via electroporation.
• * Characterization of induced DNA deletions using long-range PCR, TOPO cloning, Sanger sequencing, and next-generation sequencing.

Main Results:

• * Efficient induction of large genomic deletions (1-100 kb) in human cells using CRISPR-Cas3.
• * Successful purification and delivery of functional CRISPR-Cas3 components.
• * Characterization confirmed the precise and extensive nature of the induced deletions.

Conclusions:

• * CRISPR-Cas3 is a powerful tool for creating large genomic deletions in human cells, overcoming limitations of other CRISPR systems.
• * The provided protocols facilitate the application of CRISPR-Cas3 in human pluripotent stem cells and other cell types.
• * This technology holds significant promise for treating genetic diseases, studying gene function, and advancing genomic research.