International journal of biological macromoleculesSep 10, 2026

Using modified donor DNA improves CRISPR-Cas9 gene insertion for creating a chemical-based protein reduction system

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Sep 11, 2026

Abstract

Increased efficiency of gene knockin was achieved using donor DNA with additional gRNA-recognizable spacer sequences.

The addition of spacers to donor templates positively influenced the efficiency of homology-directed repair during CRISPR-Cas9 editing.
Systematic analysis indicated that the position, length, and orientation of spacers significantly affected HDR efficiency across various genomic loci.
Spacer-containing donors formed stable complexes with the Cas9/gRNA complex and showed enhanced nuclear localization.
This gene-editing strategy was successfully applied to create a chemical knockdown platform for rapid depletion of specific proteins in cell lines.
The new platform allows for selective modulation of endogenous proteins, revealing potentially different cellular responses compared to traditional genetic methods.

Simplified

Precise installation of functional protein domains at endogenous loci is a powerful approach for interrogating protein functions, but its broad application is limited by the low efficiency of homology-directed repair (HDR)-mediated knockin during Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene editing. Here, we investigated a simple donor DNA engineering strategy that enhances HDR-mediated gene knockin by appending additional gRNA-recognizable spacer sequences to donor templates. Systematic analysis of linear dsDNA and plasmid donors showed that spacer position, length, and orientation influenced HDR efficiency, and that spacer-containing donors improved knockin across multiple genomic loci, insertion sizes, cell types and delivery modalities. Mechanistic analyses revealed that spacer-containing donors formed stable complexes with Cas9/gRNA and showed increased nuclear localization, supporting nuclear delivery as a key contributor to improved editing outcomes. We then applied this gene-editing strategy to establish a chemical knockdown platform by installing drug-responsive degrons at endogenous loci, generating cell lines in which GSK3β or Lin28A protein could be rapidly, potently and reversibly depleted by drug treatment. These platforms enable selective modulation of endogenous proteins and reveal cellular responses that may differ from those obtained using conventional genetic perturbation. Together, this work establishes a readily implementable framework that integrates improved gene editing with on-demand chemical knockdown of endogenous proteins.

Full Text

Full text is available at the source.

View Full Text ↗

Using modified donor DNA improves CRISPR-Cas9 gene insertion for creating a chemical-based protein reduction system

Abstract

Full Text

You found one interesting study. We’ll send the next 7.

What Lands in Your Inbox Each Week:

Recent issues from the crispr gene editing brief