Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells

Area of Science:

• Stem cell biology
• Gene editing technologies
• Molecular genetics

Background:

• Efficient gene knockout is crucial for loss-of-function studies in human pluripotent stem cells (hPSCs).
• Existing methods may lack efficiency or predictability in gene ablation.
• Developing novel strategies is essential for advancing hPSC research.

Purpose of the Study:

• To introduce a novel, donor-free paired guide RNA (gRNA)-guided CRISPR/Cas9 knockout strategy (paired-KO).
• To demonstrate the efficiency and robustness of paired-KO for gene ablation in hPSCs.
• To establish a predictable method for creating gene lesions for functional studies.

Main Methods:

• Utilized a paired gRNA-guided CRISPR/Cas9 system without a DNA donor template.
• Applied the paired-KO strategy for targeting various genes, including microRNAs and long non-coding RNAs.
• Analyzed DNA repair mechanisms, focusing on direct end joining (precise ligation).

Main Results:

• Achieved high biallelic targeting efficiencies for all tested genes.
• Observed predominant DNA repair via direct end joining, resulting in predictable lesions.
• Demonstrated high efficiency for one-step multiplex gene targeting.
• Successfully targeted microRNAs and long non-coding RNAs (lncRNAs) by cleaving promoter regions.

Conclusions:

• The paired-KO strategy offers a simple, rapid, and robust method for gene ablation in hPSCs.
• This approach facilitates efficient loss-of-function studies for both coding and non-coding genes.
• Paired-KO enhances predictability in gene editing outcomes, simplifying downstream analysis.