CRISPR Replaces DNA in Non-Dividing Cells

REPLACE uses two guide RNAs to direct SpCas9 to either side of a selected genomic region. After the intervening sequence is excised, a double-stranded donor without homology arms is inserted through non-homologous end joining. Engineered virus-like particles delivered Cas9-guide RNA complexes, while adeno-associated viruses supplied the donor DNA.

The method replaced Actb exons with a reporter in approximately 35 per cent of primary mouse hepatocytes and 20 per cent of hepatocytes in vivo. In mouse zygotes, it replaced a 27-kb Ace2 region with the human ACE2 coding sequence in two of 64 offspring; the allele was transmitted through the germline and retained tissue-specific expression.

In post-mitotic cardiomyocytes derived from patient iPSCs, REPLACE corrected a frameshift mutation in LMNA in 13 per cent of long sequencing reads and restored Lamin A/C expression. However, 41 per cent of reads contained an inverted endogenous exon, 17 per cent carried a deletion and 5.5 per cent contained AAV backbone sequence. One predicted off-target site showed 1.8 per cent editing. Genome-wide off-target and chromosomal-rearrangement analyses remain necessary, and the work has not been peer-reviewed.

The study was led by Tu Dang, Alexandra Roman and Ralf Kühn at the Max Delbrück Center for Molecular Medicine. It was published in bioRxiv on 24 July 2026.