Multiplex CRISPR/Cas9-based genome engineering enhanced by Drosha-mediated sgRNA-shRNA structure

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is a powerful genome-editing tool.
• CRISPR technology relies on the Cas9 protein and single-guide RNA (sgRNA) for its function.
• Existing CRISPR methods enable multiplex targeting and homology-directed repair (HDR) in various organisms.

Purpose of the Study:

• To develop a novel strategy for multiplex genome targeting and precise genome editing using CRISPR.
• To investigate the efficacy of a Drosha-mediated sgRNA-shRNA structure for directing Cas9.
• To enhance homology-directed repair (HDR)-based genome editing efficiency.

Main Methods:

• Utilized a Drosha-mediated sgRNA-shRNA construct to direct Cas9 for genome editing.
• Performed multiplex genome targeting assays on three genomic loci.
• Introduced short hairpin RNA (shRNA) targeting the DNA ligase IV gene (LIG4) to modulate HDR efficiency.

Main Results:

• Achieved over 9% simultaneous mutant efficiency for three genomic loci in puromycin-selected cell clones.
• Demonstrated a more than 2-fold improvement in HDR-based precise genome editing efficiency by incorporating LIG4 shRNA.
• Successfully employed the novel sgRNA-shRNA construct for multiplex and precise genome modification.

Conclusions:

• The Drosha-mediated sgRNA-shRNA construct is an effective tool for multiplex CRISPR genome targeting.
• This strategy significantly enhances homology-directed repair efficiency for precise genome editing.
• The developed method offers a valuable approach for complex genome engineering in mammalian cells.