CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
17 08 2020
17 08 2020
Historique:
received:
23
05
2019
accepted:
23
07
2020
entrez:
19
8
2020
pubmed:
19
8
2020
medline:
9
9
2020
Statut:
epublish
Résumé
Precise genome editing using CRISPR-Cas9 is a promising therapeutic avenue for genetic diseases, although off-target editing remains a significant safety concern. Guide RNAs shorter than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity. Here we describe CRISPR Guide RNA Assisted Reduction of Damage (CRISPR GUARD) as a method for protecting off-targets sites by co-delivery of short guide RNAs directed against off-target loci by competition with the on-target guide RNA. CRISPR GUARD reduces off-target mutagenesis while retaining on-target editing efficiencies with Cas9 and base editor. However, we discover that short guide RNAs can also support base editing if they contain cytosines within the deaminase activity window. We explore design rules and the universality of this method through in vitro studies and high-throughput screening, revealing CRISPR GUARD as a rapidly implementable strategy to improve the specificity of genome editing for most genomic loci. Finally, we create an online tool for CRISPR GUARD design.
Identifiants
pubmed: 32807781
doi: 10.1038/s41467-020-17952-5
pii: 10.1038/s41467-020-17952-5
pmc: PMC7431537
doi:
Substances chimiques
RNA, Guide
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
4132Subventions
Organisme : Wellcome Trust
Pays : United Kingdom
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