CATI: an efficient gene integration method for rodent and primate embryos by MMEJ suppression

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Homology-directed repair (HDR) is vital for gene therapy and creating animal models.
• Microhomology-mediated end-joining (MMEJ) is a significant DNA repair pathway during CRISPR-mediated gene editing.
• MMEJ can compete with HDR, reducing the efficiency of targeted gene modifications.

Purpose of the Study:

• To investigate the role of MMEJ in CRISPR-mediated gene editing.
• To develop a method to enhance HDR efficiency by suppressing MMEJ.
• To evaluate the efficacy of the new method in mammalian embryos.

Main Methods:

• Utilized CasRx to downregulate the MMEJ factor Polymerase Q (Polq).
• Assessed the impact of Polq downregulation on the targeted integration of DNA fragments and single-strand oligonucleotides (ssODN).
• Applied the CasRX-assisted targeted integration (CATI) method in mouse and monkey embryos.

Main Results:

• Downregulation of Polq significantly improved the efficiency of targeted DNA integration in mouse embryos.
• CasRX-assisted targeted integration (CATI) demonstrated substantial improvements in HDR efficiency in monkey embryos.
• The CATI method proved effective for both linearized DNA fragments and ssODN integration.

Conclusions:

• MMEJ is a major competing pathway to HDR during CRISPR gene editing.
• CasRX-mediated downregulation of Polq is an effective strategy to enhance HDR efficiency.
• CATI offers a promising tool for generating advanced animal models and advancing gene therapies.