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Cas9‐Rep fusion tethers donor DNA in vivo and boosts the efficiency of HDR‐mediated genome editing
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Cas9‐Rep fusion tethers donor DNA in vivo and boosts the efficiency of HDR‐mediated genome editing
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Journal Article
Cas9‐Rep fusion tethers donor DNA in vivo and boosts the efficiency of HDR‐mediated genome editing
2025
Overview
Summary Genome editing based on the homology‐directed repair (HDR) pathway enables scar‐free and precise genetic manipulations. However, the low frequency of HDR hinders its application in plant genome editing. In this study, we engineered the fusion of Cas9 and a viral replication protein (Rep) as a molecular bridge to tether donor DNA in vivo, which enhances the efficiency of targeted gene insertion via the HDR pathway. This Rep‐bridged knock‐in (RBKI) method combines the advantages of rolling cycle replication of viral replicons and in vivo enrichment of donor DNA at the target site for HDR. Chromatin immunoprecipitation indicated that the Cas9‐Rep fusion protein bound up to 66‐fold more donor DNA than Cas9 did. We exemplified the RBKI method by inserting small‐ to middle‐sized tags (33–519 bp) into 3 rice genes. Compared to Cas9, Cas9‐Rep fusion increased the KI frequencies by 4–7.6‐fold, and up to 72.2% of stable rice transformants carried in‐frame knock‐in events in the T0 generation. Whole‐genome sequencing of 6 plants segregated from heterozygous KI lines indicated that the knock‐in events were faithfully inherited by the progenies with neither off‐target editing nor random insertions of the donor DNA fragment. Further analysis suggested that the RBKI method reduced the number of byproducts from nonhomologous end joining; however, HDR‐mediated knock‐in tended to accompany microhomology‐mediated end joining events. Together, these findings show that the in vivo tethering of donor DNAs with Cas9‐Rep is an effective strategy to increase the frequency of HDR‐mediated genome editing.
Publisher
John Wiley & Sons, Inc,John Wiley and Sons Inc
Subject
/ chromatin immunoprecipitation
/ CRISPR
/ CRISPR-Associated Protein 9 - genetics
/ CRISPR-Associated Protein 9 - metabolism
/ CRISPR-Cas Systems - genetics
/ DNA
/ Editing
/ genes
/ Genomes
/ Homology
/ Kinases
/ knock‐in
/ Plants, Genetically Modified
/ Proteins
/ Recombinational DNA Repair - genetics
/ Rep
/ rice
/ Tethers
