An optimized SpCas9 high-fidelity variant for direct protein delivery.
CRISPR-Cas
HDR
RNP
genome editing
hematopoietic stem cells
high-fidelity Cas9
protein delivery
specificity
Journal
Molecular therapy : the journal of the American Society of Gene Therapy
ISSN: 1525-0024
Titre abrégé: Mol Ther
Pays: United States
ID NLM: 100890581
Informations de publication
Date de publication:
05 07 2023
05 07 2023
Historique:
received:
08
12
2022
revised:
08
02
2023
accepted:
07
03
2023
pmc-release:
05
07
2024
medline:
10
7
2023
pubmed:
12
3
2023
entrez:
11
3
2023
Statut:
ppublish
Résumé
Electroporation of the Cas9 ribonucleoprotein (RNP) complex offers the advantage of preventing off-target cleavages and potential immune responses produced by long-term expression of the nuclease. Nevertheless, the majority of engineered high-fidelity Streptococcus pyogenes Cas9 (SpCas9) variants are less active than the wild-type enzyme and are not compatible with RNP delivery. Building on our previous studies on evoCas9, we developed a high-fidelity SpCas9 variant suitable for RNP delivery. The editing efficacy and precision of the recombinant high-fidelity Cas9 (rCas9HF), characterized by the K526D substitution, was compared with the R691A mutant (HiFi Cas9), which is currently the only available high-fidelity Cas9 that can be used as an RNP. The comparative analysis was extended to gene substitution experiments where the two high fidelities were used in combination with a DNA donor template, generating different ratios of non-homologous end joining (NHEJ) versus homology-directed repair (HDR) for precise editing. The analyses revealed a heterogeneous efficacy and precision indicating different targeting capabilities between the two variants throughout the genome. The development of rCas9HF, characterized by an editing profile diverse from the currently used HiFi Cas9 in RNP electroporation, increases the genome editing solutions for the highest precision and efficient applications.
Identifiants
pubmed: 36905119
pii: S1525-0016(23)00128-4
doi: 10.1016/j.ymthe.2023.03.007
pmc: PMC10362380
pii:
doi:
Substances chimiques
CRISPR-Associated Protein 9
EC 3.1.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
2257-2265Informations de copyright
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of interests A. Casini and A. Cereseto are founders of and hold shares in Alia Therapeutics, a genome editing company. A. Casini is an employee of Alia Therapeutics, and A. Cereseto is a consultant for Alia Therapeutics. A patent application covering the technology disclosed in this manuscript has been filed, and A. Casini and A. Cereseto are listed as inventors.
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