1.7kb Determines the Success Rate of Gene Editing

Introduction

Since the development of gene editing technology, precise targeted integration has always been the "holy grail" pursued by researchers. Recently, the team of Noritaka Adachi from Yokohama City University published an important discovery in PNAS: "Homology- arm length of donor DNA affects the impact of Msh2 loss on homologous recombination-mediated gene targeting". This study revealed a key factor affecting gene targeting efficiency - the subtle relationship between the length of the homology arm of donor DNA and the mismatch repair protein Msh2.

Unexpected Discovery: Hidden Obstacles of Short-arm Vectors

The research team discovered an interesting phenomenon during the gene targeting experiment: when using donor DNA with the same gene sequence for targeted integration, the presence or absence of Msh2 protein will produce completely different effects, and this difference actually depends on the length of the homology arm.

Specifically, when the researchers compared two targeting vectors of different lengths:

• Long homology arm vector (p8.9HPRT-Puro, total length 8.9kb): Msh2 deletion had almost no effect on targeting efficiency.
• Short homology arm vector (p3.0HPRT-Puro, total length 3.0kb): Msh2 deletion increased targeting efficiency by about 6 times.

Critical Threshold: 1.7kb Watershed

To further confirm this finding, the research team constructed multiple targeting vectors of different lengths for systematic analysis. The results revealed an important rule: When a single homology arm is as short as 1.7kb or shorter, the inhibitory effect of Msh2 will be apparent. This finding breaks the previous belief that "homologous DNA is not affected by mismatch repair".

More importantly, the data revealed a clear negative correlation between the total homology arm length and the degree of Msh2 influence - the shorter the homology arm, the more obvious the efficiency improvement brought by Msh2 deficiency.

Mechanism Deciphering: Two Different Targeting Pathways

The study further found that gene targeting can be achieved through two different mechanisms.

• Homologous recombination (HR) pathway: dependent on Rad51 protein, affected by Msh2 and related to homology arm length.
• Single-strand annealing (SSA) pathway: dependent on Rad52 protein, less affected by Msh2 and unrelated to homology arm length.

This finding explains why the effect of Msh2 becomes independent of homology arm length in HR-deficient cells.

A Major Breakthrough in Gene Editing Applications

When Msh2, LIG4 and POLQ were inhibited simultaneously, the targeting efficiency of the short homology arm vector was increased by 20 times! This discovery is of great significance to gene editing applications.

Optimizing Vector Design Strategies

• For cell lines with normal mismatch repair function, it is recommended to use long homology arm vectors to obtain efficient targeting.
• For cell lines with mismatch repair defects, short homology arm vectors are also effective.

A New Strategy to Improve Gene Editing Efficiency

The study proposed an innovative combination strategy: Msh2 inhibition + NHEJ inhibition + short homology arm vector, which can significantly improve the efficiency of targeted integration. This is of special value for gene editing systems that are limited by vector length (such as rAAV vectors, the total length cannot exceed 4kb).

Rethinking Cell Line Selection

When performing gene editing, the mismatch repair status of the cell line should be taken into consideration, which not only affects vector design, but also affects the formulation of experimental strategies.

Theoretical Model: Interference Effect of DNA End Resection

The research team proposed an explanatory model. During homologous recombination, when DNA double-strand breaks undergo end resection, short homologous arms may not provide sufficiently long homologous sequences to overcome the interference of Msh2 on non-homologous sequences (such as marker genes). This is highly consistent with the known average length of DNA end resection (about 1-2kb).

Figure 1. Model for Msh2-mediated suppression of TI with a short-arm targeting vector. (Saito, et al., 2025)

Summary and Outlook

This study not only reveals a new mechanism of gene targeting, but also provides a practical strategy to improve gene editing efficiency. In particular, for gene therapy applications that require the use of short vectors, this discovery may bring revolutionary improvements.

In the future, combined with modern gene editing tools such as CRISPR/Cas9, through a combination of reasonable cell line selection, vector design and protein inhibition strategies, we are expected to achieve more efficient and precise gene editing, opening up new possibilities for gene therapy and biomedical research.

Agrobacterium Competent Cells We Offer

Cat#	Product Name	Size
ACC-100	GV3101 Chemically Competent Cell	10 tubes (100μL/tube) 20 tubes (100μL/tube) 50 tubes (100μL/tube) 100 tubes (100μL/tube)
ACC-103	EHA105 Chemically Competent Cell	10 tubes (100μL/tube) 20 tubes (100μL/tube) 50 tubes (100μL/tube) 100 tubes (100μL/tube)
ACC-105	AGL1 Chemically Competent Cell	10 tubes (100μL/tube) 20 tubes (100μL/tube) 50 tubes (100μL/tube) 100 tubes (100μL/tube)
ACC-107	LBA4404 Chemically Competent Cell	10 tubes (100μL/tube) 20 tubes (100μL/tube) 50 tubes (100μL/tube) 100 tubes (100μL/tube)
ACC-108	EHA101 Chemically Competent Cell	10 tubes (100μL/tube) 20 tubes (100μL/tube) 50 tubes (100μL/tube) 100 tubes (100μL/tube)
ACC-117	Ar.Qual Chemically Competent Cell	10 tubes (100μL/tube) 20 tubes (100μL/tube) 50 tubes (100μL/tube) 100 tubes (100μL/tube)
ACC-118	MSU440 Chemically Competent Cell	10 tubes (100μL/tube) 20 tubes (100μL/tube) 50 tubes (100μL/tube) 100 tubes (100μL/tube)
ACC-119	C58C1 Chemically Competent Cell	10 tubes (100μL/tube) 20 tubes (100μL/tube) 50 tubes (100μL/tube) 100 tubes (100μL/tube)
ACC-121	K599 Chemically Competent Cell	10 tubes (100μL/tube) 20 tubes (100μL/tube) 50 tubes (100μL/tube) 100 tubes (100μL/tube)
ACC-122	Ar.A4 Electroporation Competent Cell	10 tubes (50μL/tube) 20 tubes (50μL/tube) 50 tubes (50μL/tube)

Related Services & Products

• Plant Genetic Engineering
• CRISPR/CAS9 Service
• Gene Overexpression Service
• RNAi Service
• CRISPR/Cas12a Service
• CRISPR Base Editors Service
• CRISPRa Service
• CRISPR/Cas12b Service
• CRISPRoff/CRISPRon Service
• CRISPR Interference (CRISPRi) Service
• Fish Gene Editing
• Poultry, Livestock, Pets and Laboratory Animal Gene Editing
• Insect Gene Editing
• Vectors

Reference

1. Saito, S., et al. (2025). Homology-arm length of donor DNA affects the impact of Msh2 loss on homologous recombination–mediated gene targeting. Proceedings of the National Academy of Sciences, 122(24), e2508507122. DOI: 1073/pnas.2508507122.