Articolo in rivista, 2023, ENG, 10.1016/j.ymthe.2023.03.007

An optimized SpCas9 high-fidelity variant for direct protein delivery

Eleonora Pedrazzoli; Andrea Bianchi; Alessandro Umbach; Simone Amistadi; Mégane Brusson; Giacomo Frati; Matteo Ciciani; Kalina Aleksandra Badowska; Daniele Arosio; Annarita Miccio; Anna Cereseto and Antonio Casini.

Department CIBIO, Laboratory of Molecular Virology, University of Trento, Via Sommarive 9, 38123 Trento, Italy; Imagine Institute, Laboratory of Chromatin and Gene Regulation During Development, Université de Paris, INSERM UMR 1163, Paris, France; Alia Therapeutics, 38123 Trento, Italy; Biophysics Institute, National Research Council of Italy, 38123 Trento, Italy

Electroporation of the Cas9 ribonucleoprotein (RNP) complex offers the advantage of preventing off-target cleavages and po- tential immune responses produced by long-term expression of the nuclease. Nevertheless, the majority of engineered high-fi- delity 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 sub- stitution, 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 var- iants 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.

Molecular therapy (Online) 31 (7), pp. 2257–2265

Keywords

CRISPR-Cas; protein delivery; RNP HDR; hematopoietic stem cells; high-fidelity; Cas9 specificity; genome editing

CNR authors

Arosio Daniele

CNR institutes

IBF – Istituto di biofisica

ID: 484294

Year: 2023

Type: Articolo in rivista

Creation: 2023-07-12 11:33:54.000

Last update: 2023-07-12 11:33:54.000

CNR authors

External links

OAI-PMH: Dublin Core

OAI-PMH: Mods

OAI-PMH: RDF

DOI: 10.1016/j.ymthe.2023.03.007

External IDs

CNR OAI-PMH: oai:it.cnr:prodotti:484294

DOI: 10.1016/j.ymthe.2023.03.007