![[feed]](/style/images/feed-icon-14x14.png){kind=icon}
Tools
Tools
| Item Type: | Preprint |
|---|---|
| Title: | Efficient CRISPR/Cas9-mediated homology independent sequence replacement in vivo and non-dividing primary cells |
| Creators: |
Dang, Tu Ngoc  ORCID: https://orcid.org/0009-0001-0138-7599, Roman, Alexandra ORCID: https://orcid.org/0000-0001-9440-9752, Zimmer, Anja, Lebedin, Mikhail ORCID: https://orcid.org/0000-0002-3877-7195, Bahry, Ella ORCID: https://orcid.org/0000-0002-6358-2576, Grifol, Claudia Marti ORCID: https://orcid.org/0009-0004-9929-0585, Esser, Markus, Sevim Wunderlich, Seren ORCID: https://orcid.org/0009-0008-2771-4149, Miller, Duncan C. ORCID: https://orcid.org/0000-0001-9852-677X, Diecke, Sebastian ORCID: https://orcid.org/0000-0002-5219-5992 and Kuehn, Ralf ORCID: https://orcid.org/0000-0003-1694-9803
|
| Abstract: | Precise sequence replacement in non-dividing cells remains a major challenge for genome editing. Here we show that REPLACE (Rational end-joining protocol delivering a targeted sequence exchange), a homology-independent CRISPR/Cas9-based replacement strategy, enables exon- and gene-scale substitution in primary cells, in vivo tissues and post-mitotic human cardiomyocytes. REPLACE uses two guide RNAs to excise a defined genomic region and inserts a donor lacking homology arms through non-homologous end joining (NHEJ). In primary mouse hepatocytes, REPLACE mediated exon replacement in 35% of all cells. In adult mouse liver, editing efficiency could be increased to ~20% when Cas9-sgRNA ribonucleoproteins were delivered via engineered virus-like particles (eVLPs) together with an adeno-associated virus (AAV) donor. REPLACE also supported large-segment replacement, enabling one-step exchange of a ~27-kb mouse Ace2 interval with the human ACE2 coding region in zygotes, followed by germline transmission and tissue-specific expression. Finally, we applied REPLACE to a disease-relevant mutation that is not readily addressable by base editing and was poorly corrected by prime editing in post-mitotic cardiomyocytes. At the LMNA locus, REPLACE corrected the K117fs frameshift mutation in patient-derived post-mitotic cardiomyocytes with precise exon replacement and restored Lamin A/C protein expression and nuclear lamina localization. These findings establish REPLACE as a versatile platform for homology-independent sequence replacement and as a complementary approach for genetic correction in settings where homology-directed repair (HDR), base editing (BE) or prime editing (PE) are inefficient or not applicable. |
| Keywords: | Animals, Mice |
| Source: | bioRxiv |
| Publisher: | Cold Spring Harbor Laboratory Press |
| Article Number: | 2026.07.24.740048 |
| Date: | 24 July 2026 |
| Additional Information: | The following ID is not public in BioProject: 1366436. |
| Official Publication: | https://doi.org/10.64898/2026.07.24.740048 |
| Related to: |
Repository Staff Only: item control page
