Modulation of the microhomology-mediated end joining pathway suppresses large deletions and enhances homology-directed repair following CRISPR-Cas9-induced DNA breaks.
CRISPR-Cas9 genome editing
Homology-directed repair
Large deletions
Microhomology-mediated end joining
Journal
BMC biology
ISSN: 1741-7007
Titre abrégé: BMC Biol
Pays: England
ID NLM: 101190720
Informations de publication
Date de publication:
29 Apr 2024
29 Apr 2024
Historique:
received:
12
06
2023
accepted:
18
04
2024
medline:
30
4
2024
pubmed:
30
4
2024
entrez:
29
4
2024
Statut:
epublish
Résumé
CRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks. Using long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells. Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing.
Sections du résumé
BACKGROUND
BACKGROUND
CRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks.
RESULTS
RESULTS
Using long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells.
CONCLUSIONS
CONCLUSIONS
Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing.
Identifiants
pubmed: 38685010
doi: 10.1186/s12915-024-01896-z
pii: 10.1186/s12915-024-01896-z
doi:
Substances chimiques
DNA Polymerase theta
EC 2.7.7.-
Replication Protein A
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
101Subventions
Organisme : King Abdullah University of Science and Technology
ID : BAS/1/1080-01
Informations de copyright
© 2024. The Author(s).
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