BMC plant biologyOct 1, 2025

Improved targeted gene editing in soybean using a CRISPR system combined with an exonuclease

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Jun 27, 2026

Abstract

Essence

-fused CRISPR systems broaden soybean genome editing by shifting repair outcomes toward larger targeted deletions.

Evidence

A soybean platform experiment at the GmWOX5 locus used Agrobacterium rhizogenes transformation and deep amplicon sequencing to compare native Cas9/Cas12a with T5-Exo and TREX2 fusions, which increased moderate-to-large deletion classes and reduced insertions for Cas9 fusions.

Caveat

The abstract reports editing-spectrum data at one soybean locus, so performance across other targets, traits, and whole-plant applications remains uncertain.

Simplified

technologies have revolutionized plant genome editing, yet their inherent bias toward small insertions or deletions (indels) limits their utility for dissecting regulatory elements and generating impactful allelic variants. Here, we report the development and systematic evaluation of -fused CRISPR/Cas systems in soybean to overcome this limitation. We engineered fusions of Cas9 and Cas12a with bacteriophage T5 exonuclease and human TREX2 and assessed their editing performance at the GmWOX5 locus using Agrobacterium rhizogenes-mediated transformation and deep amplicon sequencing. While native Cas9 and Cas12a predominantly generated micro-size deletions (1-10 bp), T5-Exo fusions shifted the mutation spectrum, producing a high frequency of moderate (26-50 bp) and large (> 50 bp) deletions. TREX2 fusions preferentially enhanced the generation of small (11-25 bp) to moderate deletions (26-50 bp). Fusion of exonucleases to Cas9 substantially reduced insertion frequencies and promoted more precise deletion patterns, as observed in T5-Exo-Cas9 and TREX2-Cas9. Deletions from both exonuclease fusions were biased toward the PAM-proximal region, reflecting altered repair outcomes likely driven by directional exonuclease activity and enhanced end resection. These results demonstrate that exonuclease fusions effectively expand the CRISPR toolkit by enabling efficient, targeted generation of larger deletions, which are often required for targeting cis-regulatory elements and microRNAs.

Key numbers

27%

Moderate Deletions Frequency Increase

T5 Exo- generated 27% moderate deletions (26–50 bp).

84%

Microdeletions by

predominantly produced microdeletions (1–10 bp) at 84%.

67%

-Proximal Deletions

67% of deletions from T5 Exo- occurred at the -proximal end.

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Improved targeted gene editing in soybean using a CRISPR system combined with an exonuclease

Abstract

Key numbers

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