Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases.
Nat Commun
; 12(1): 4219, 2021 07 09.
Article
en En
| MEDLINE
| ID: mdl-34244505
ABSTRACT
Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Staphylococcus
/
Sistemas CRISPR-Cas
/
Edición Génica
/
Proteína 9 Asociada a CRISPR
Límite:
Animals
/
Humans
/
Male
Idioma:
En
Revista:
Nat Commun
Asunto de la revista:
BIOLOGIA
/
CIENCIA
Año:
2021
Tipo del documento:
Article
País de afiliación:
Alemania