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Nat Biotechnol. 2018 Sep;36(8):765-771. doi: 10.1038/nbt.4192. Epub 2018 Jul 16.

Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements.

Author information

1
Wellcome Sanger Institute, Hinxton, UK.

Abstract

CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.

PMID:
30010673
DOI:
10.1038/nbt.4192

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