Target-enriched nanopore sequencing and de novo assembly reveals co-occurrences of complex on-target genomic rearrangements induced by CRISPR-Cas9 in human cells.
Journal
Genome research
ISSN: 1549-5469
Titre abrégé: Genome Res
Pays: United States
ID NLM: 9518021
Informations de publication
Date de publication:
10 2022
10 2022
Historique:
received:
05
05
2022
accepted:
19
09
2022
pubmed:
1
10
2022
medline:
5
11
2022
entrez:
30
9
2022
Statut:
ppublish
Résumé
The CRISPR-Cas9 system is widely used to permanently delete genomic regions via dual guide RNAs. Genomic rearrangements induced by CRISPR-Cas9 can occur, but continuous technical developments make it possible to characterize complex on-target effects. We combined an innovative droplet-based target enrichment approach with long-read sequencing and coupled it to a customized de novo sequence assembly. This approach enabled us to dissect the sequence content at kilobase scale within an on-target genomic locus. We here describe extensive genomic disruptions by Cas9, involving the allelic co-occurrence of a genomic duplication and inversion of the target region, as well as integrations of exogenous DNA and clustered interchromosomal DNA fragment rearrangements. Furthermore, we found that these genomic alterations led to functional aberrant DNA fragments and can alter cell proliferation. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations, and introduce a data-driven workflow enabling detailed dissection of the on-target sequence content with superior resolution.
Identifiants
pubmed: 36180232
pii: gr.276901.122
doi: 10.1101/gr.276901.122
pmc: PMC9712622
doi:
Substances chimiques
RNA, Guide
0
DNA
9007-49-2
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1876-1891Informations de copyright
© 2022 Geng et al.; Published by Cold Spring Harbor Laboratory Press.
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