CRISPR RNAs trigger innate immune responses in human cells.
Genome Res
; 2018 02 22.
Article
em En
| MEDLINE
| ID: mdl-29472270
ABSTRACT
Here, we report that CRISPR guide RNAs (gRNAs) with a 5'-triphosphate group (5'-ppp gRNAs) produced via in vitro transcription trigger RNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5'-ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to â¼80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting 5'-hydroxyl gRNAs in complex with Cas9 or Cpf1 avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4+ T cells. These results are in line with previous findings that chemically synthesized sgRNAs with a 5'-hydroxyl group are much more efficient than in vitro-transcribed (IVT) sgRNAs in human and other mammalian cells. The phosphatase treatment of IVT sgRNAs is a cost-effective method for making highly active sgRNAs, avoiding innate immune responses in human cells.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Idioma:
En
Revista:
Genome Res
Assunto da revista:
BIOLOGIA MOLECULAR
/
GENETICA
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Coréia do Sul