Nature communicationsDec 9, 2025

High-precision gene insertion using recombinase-enhanced CRISPR-Cas9

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

Updated Jun 27, 2026

Abstract

Essence

RED-CRISPR may improve precise kilobase-scale DNA knock-in across cell and mouse genome-editing systems.

Evidence

This genome-editing platform experiment tested immortalized and primary cells, CAR-T manufacturing, and genetically modified mice, reporting 2- to 5-fold higher HDR, up to 45% CAR-T knock-in, and 43% mouse knock-in with an 8-kb cargo.

Caveat

The results come from engineered preclinical systems, so broad biomedical or therapeutic usefulness remains inferred rather than clinically shown.

Simplified

CRISPR-Cas9 tools have revolutionized genetic engineering, yet the efficient precise integration of DNA cargos, particularly for large DNA payloads (>1 kilobase, kb), remains a technical bottleneck. Herein, we develop a Recombinases (Redα/β)-enhanced DNA integration-CRISPR-Cas9 approach, referred to as RED-CRISPR, which offers a versatile yet robust homology-directed repair (HDR) strategy enabling efficient and precise kb-scale DNA insertion across various cell types, including immortalized and primary cells of variable origins. RED-CRISPR significantly enhances HDR efficiencies by 2- to 5-fold change across diverse loci and further elevates HDR rates by 1.5- to 2.5-fold when synergizing with other HDR-enhancing strategies. We achieved up to 45% knock-in efficiency for CAR-T cell manufacturing, and attained 43% knock-in rate for generation of genetically modified mice using an 8-kb DNA cargo. Through a head-to-head comparison, RED-CRISPR profoundly mitigates off-target mutational burden and chromosomal translocations. We envision RED-CRISPR as a powerful genome-editing tool with broad biomedical and therapeutic applications.