Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-based gene editing typically uses non-homologous end-joining (NHEJ) for gene knock-outs.
• Homology-directed repair (HDR) allows for precise gene insertion/deletion but is often inefficient for large fragments.
• Previous studies reported limitations in knocking in DNA fragments larger than 4 kb via HDR.

Purpose of the Study:

• To demonstrate the efficient targeted insertion of large DNA fragments (7.4 and 5.8 kb) using CRISPR/HDR.
• To investigate the efficacy of CRISPR/HDR without NHEJ inhibitors for large fragment knock-in.
• To assess the occurrence of NHEJ and biallelic gene knock-ins during large fragment knock-in via CRISPR/HDR.

Main Methods:

• Utilized CRISPR/HDR technique for targeted gene knock-in of large DNA fragments (7.4 kb and 5.8 kb).
• Performed experiments in mouse embryonic stem (ES) cells and zygotes.
• Did not employ NHEJ inhibitors during the CRISPR/HDR process.

Main Results:

• Achieved highly efficient gene knock-in of large DNA fragments (7.4 and 5.8 kb) using CRISPR/HDR without NHEJ inhibitors.
• Demonstrated knock-in efficiency comparable to methods using NHEJ inhibitors.
• Observed no detectable NHEJ or biallelic gene knock-ins, despite NHEJ being the dominant repair pathway for CRISPR-induced double-strand breaks.

Conclusions:

• Efficient targeted insertion of large DNA fragments (>4 kb) is achievable with CRISPR/HDR without NHEJ inhibitors.
• This method offers a viable alternative for precise large fragment knock-in in genome engineering.
• Findings encourage further research into the mechanisms underlying high-efficiency CRISPR targeting.