Cas3-mediated Heritable Large Gene Editing

On June 6, 2025, Seiichi Toki's team from the Japan Agricultural and Food Research Institute published a new research paper titled "Heritable Large Deletions Using Type I-E CRISPR-Cas3 in Rice" on bioRxiv. This study successfully achieved heritable large gene deletion editing mediated by Type I-E CRISPR-Cas3 in rice for the first time. This breakthrough has added an important new member to the plant gene editing toolbox.

Technical Difficulties Faced by Cas3 Editing

Compared with the familiar CRISPR-Cas9 system, the application of Type I CRISPR-Cas system in plants has always faced great challenges. The research team pointed out that the Type I-E CRISPR-Cas3 system requires the simultaneous expression of 7 components (6 Cas proteins and CRISPR RNA), which makes its application in plant cells extremely difficult.

Previously, the application of Cas3 in plants was limited to corn protoplasts, and mutant plants had never been obtained. This has become an important bottleneck restricting the promotion and application of this technology.

Innovative Strategies to Overcome Technical Difficulties

To overcome these difficulties, the research team developed a clever combination of strategies.

Eco CRISPR-Cas3-mediated targeted mutagenesis in rice calli. A. Eco CRISPR-Cas3 vector targeting OsPDS; B. OsPDS locus structure; C. Transformation; D. PCR analysis.

Figure 1. Eco CRISPR-Cas3-mediated targeted mutagenesis in rice calli. (Saika, et al., 2025)

Innovation In Vector Design

Two vector systems were developed: using two binary vectors to carry different Cas gene expression cassettes, and creating a compact "omnipotent vector";
Six Cas gene expression cassettes were separated into two vectors: Cas537 (expressing EcoCas3, EcoCas5, and EcoCas7) and Cas6811 (expressing EcoCas6, EcoCas8, and EcoCas11).

Expression Optimization Strategy

Rice codon optimization and addition of bipartite nuclear localization signals for all Cas effector proteins.
Maize ubiquitin promoter was used to achieve strong expression.
2A self-cleavage peptide was used to fuse multiple proteins in the omnipotent vector.

Cas3 vs Cas9: Distinct Editing Characteristics

The study revealed significant differences between Cas3 and traditional Cas9 systems.

Huge Differences in Deletion Size

Cas3 introduces deletions of 0.1-7.2 kb, while Cas9 mainly produces small insertions/deletions.
Cas3 can introduce large unidirectional deletions, starting 0-400 bp upstream of the PAM sequence.

Higher Targeting Accuracy

Cas3 recognizes long target sequences of 32-37 nucleotides, significantly reducing the risk of off-target deletions compared to Cas9's 20 nucleotides.
No off-target large deletion events were detected in the study.

Unique Mutation Pattern

Sequence analysis showed that Cas3 can not only produce simple large deletions, but also create new alleles such as complex deletions separated by intervening fragments, deletions accompanied by short insertions and inversions.

Verification of Cas3 Editing Efficiency

The research team verified the editing efficiency of Cas3 by multiple methods.

Callus Level Detection

Co-transformation of 2 binary vectors: 39-48% of callus detected deletions.
All-in-one vector transformation: 55-71% of callus detected deletions.

Precise Quantitative Analysis

Using droplet digital PCR (ddPCR) technology, the researchers accurately determined the deletion frequency at different positions:

The deletion frequency of the 2.6 kb region upstream of PAM was 39.5-74.1%.
The deletion frequency of the 7.0 kb region upstream of PAM was 21.1-60.8%.
The deletion frequency of the 6.0 kb region downstream of PAM was only 1.5-25.2%, confirming the unidirectional deletion characteristics of Cas3.

Important Verification of Stable Inheritance

The most important breakthrough of the study is that it proves that large deletions mediated by Cas3 can be stably inherited by offspring.

In the OsWx gene editing experiment, the researchers obtained a T0 plant with a 2.8 kb deletion, all 12 of whose T1 seeds showed a waxy phenotype, and PCR analysis confirmed the stable inheritance of biallelic mutations.

This result shows that Cas3 can produce biallelic mutant plants with an efficiency of 14%, providing a new and powerful tool for plant breeding.

Summary

The research team pointed out that the unique large deletion characteristics of Cas3 make it a promising tool for gene knockout, gene deletion and genome rearrangement. Compared with the traditional method that requires the design of dozens of gRNAs to achieve large promoter deletions, Cas3 only needs a single gRNA to create diverse promoter alleles.

This study established a complete I-E type CRISPR-Cas3 gene editing system in plants for the first time, which not only expanded the plant gene editing toolbox, but also opened up a new technical path for crop precision improvement and molecular breeding. With the further optimization and promotion of this technology, we have reason to expect more breakthrough applications.

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)