ACS synthetic biologyMay 6, 2025

Accurate and Efficient Insertion of Large DNA Pieces Using Precise Linking of Long Single-Stranded DNA and Guide RNA

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Mar 16, 2026

Abstract

The approach achieved an unprecedented knock-in (KI) rate of approximately 36% for a gene-sized 1.4 kilobase long single-stranded DNA insertion in HEK293T cells.

A novel method named AOLP synthesizes chemically modified long single-stranded DNA (lssDNA) as a donor template for gene editing.
This method enhances the stability of lssDNA compared to conventional phosphorylation techniques.
Cyanovinylcarbazole nucleoside (K) is used for precise ligation-based interstrand cross-linking between lssDNA and sgRNA, promoting the upregulation of the homology-directed repair (HDR) pathway.
The light-activated ligation improves knock-in efficiency and reduces off-target effects associated with lssDNA donors.
The efficiency of lssDNA in K562, HEK293T, and HepG2 cells is improved by 4- to 12-fold compared to traditional methods.
The accuracy of the HDR pathway in HEK293T cells is enhanced by over 4.7-fold relative to previous commercial lssDNA.

Simplified

Homology-directed repair (HDR) allows the precise introduction of functional constructs into the human genome through nonviral gene-editing reagents. However, its application in large DNA sequence gene editing remains limited due to challenges such as low efficiency and the off-target effect. To address these limitations, a new method named AOLP was developed to synthesize chemically modified long single-stranded DNA (lssDNA) as the template donor for Cas9-based gene editing, which has been proven to be more stable than that prepared using the commercial phosphorylation method. We propose a novel strategy involving precise ligation-based interstrand cross-linking between lssDNA and sgRNA using cyanovinylcarbazole nucleoside (K), enhancing the upregulation of the HDR pathway for DSB repair induced by Cas9. The light-activated ligation between Cas9/sgRNA and lssDNA improves the knock-in (KI) efficiency, overcomes the challenges of low KI efficiency, and surpasses the low off-target effect accompanied by the lssDNA donor. Moreover, the interstrand cross-linking of lssDNA and sgRNA can subtly control the ligation sites and the degree of cross-linking of lssDNA and sgRNA to enhance the KI accuracy of HDR. Our approach improves the KI efficiency of lssDNA in K562, HEK293T, and HepG2 cells by 4- to 12-fold relative to conventional lssDNA donors prepared using the phosphorylation method. Furthermore, the KI accuracy of HDR pathway in HEK293T cells is enhanced by >4.7-fold relative to previous commercial lssDNA. Leveraging this approach, we achieved an unprecedented KI rate of approximately 36% for a gene-sized 1.4 kilobase lssDNA insertion in HEK293T cells. CNV

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Accurate and Efficient Insertion of Large DNA Pieces Using Precise Linking of Long Single-Stranded DNA and Guide RNA

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