Co-incident insertion enables high efficiency genome engineering in mouse embryonic stem cells

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9 technology offers powerful genome editing but is limited by inefficient homology-directed repair (HDR) compared to non-homologous end joining (NHEJ).
• Precise genetic modifications in mammalian genomes often require efficient HDR, which is challenging to achieve in most cell types.

Purpose of the Study:

• To enhance the efficiency of isolating precise HDR-mediated genome editing events in mouse embryonic stem cells.
• To investigate a novel method for enriching cells proficient in HDR.

Main Methods:

• Utilized co-incidental insertion (COIN) of independent donor DNA sequences to improve HDR efficiency.
• Employed selection cassettes targeting multiple loci and analyzed on:off-target frequencies.
• Tracked individual cell events using flow cytometry and fluorescent protein markers.

Main Results:

• Achieved over a 20-fold increase in isolating precise HDR-mediated events.
• Observed frequent bi-allelic insertion of selection cassettes, suggesting enrichment of HDR-proficient cells.
• Demonstrated that individual cells predominantly perform either HDR or non-homologous end joining, but not both.

Conclusions:

• COIN effectively enriches for a sub-population of HDR-proficient cells within heterogeneous cell populations.
• The COIN method, especially when combined with self-excising selection cassettes, provides highly efficient and scarless genome editing.
• This approach advances precise genome engineering capabilities in mammalian cells.