CRISPR-Cas9 HDR optimization: RAD52, denatured, and 5'-modified DNA templates in knock-in mice generation.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas9 genome editing facilitates the creation of animal disease models.
• Homology-directed repair (HDR) efficiency is a significant hurdle in precise genome editing.
• Generating conditional knockout (cKO) mouse models requires optimized HDR strategies.

Purpose of the Study:

• To investigate methods for enhancing the efficiency and precision of homology-directed repair (HDR) in CRISPR-Cas9 genome editing.
• To identify critical factors influencing precise knock-in model generation.
• To optimize the generation of a conditional knockout (cKO) mouse model for the Nup93 gene.

Main Methods:

• Injected CRISPR-Cas9 components and various DNA donor templates into over 2,000 mouse zygotes.
• Tested strategies including dsDNA template denaturation, RAD52 supplementation, and dual crRNA targeting.
• Evaluated the impact of donor DNA 5' end modifications (5'-biotin, 5'-C3 spacer) on HDR efficiency and precision.

Main Results:

• Denaturing long 5'-monophosphorylated dsDNA templates improved precise editing and reduced template multiplication.
• RAD52 supplementation increased ssDNA integration but also template multiplication.
• Targeting the antisense strand with two crRNAs enhanced HDR precision.
• 5'-biotin modification increased single-copy integration up to 8-fold; 5'-C3 spacer modification increased correctly edited mice up to 20-fold.

Conclusions:

• Specific modifications to DNA donor templates significantly enhance CRISPR-Cas9 HDR efficiency and precision.
• 5'-C3 spacer modification of donor DNA is a highly effective strategy for increasing the yield of precisely edited mice.
• These findings provide practical approaches for improving the generation of genetically engineered animal models.