Engineering CRISPR/Cpf1 with tRNA promotes genome editing capability in mammalian systems.
Cell Mol Life Sci
; 75(19): 3593-3607, 2018 Oct.
Article
em En
| MEDLINE
| ID: mdl-29637228
ABSTRACT
CRISPR/Cpf1 features a number of properties that are distinct from CRISPR/Cas9 and provides an excellent alternative to Cas9 for genome editing. To date, genome engineering by CRISPR/Cpf1 has been reported only in human cells and mouse embryos of mammalian systems and its efficiency is ultimately lower than that of Cas9 proteins from Streptococcus pyogenes. The application of CRISPR/Cpf1 for targeted mutagenesis in other animal models has not been successfully verified. In this study, we designed and optimized a guide RNA (gRNA) transcription system by inserting a transfer RNA precursor (pre-tRNA) sequence downstream of the gRNA for Cpf1, protecting gRNA from immediate digestion by 3'-to-5' exonucleases. Using this new gRNAtRNA system, genome editing, including indels, large fragment deletion and precise point mutation, was induced in mammalian systems, showing significantly higher efficiency than the original Cpf1-gRNA system. With this system, gene-modified rabbits and pigs were generated by embryo injection or somatic cell nuclear transfer (SCNT) with an efficiency comparable to that of the Cas9 gRNA system. These results demonstrated that this refined gRNAtRNA system can boost the targeting capability of CRISPR/Cpf1 toolkits.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
/
RNA de Transferência
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Clonagem Molecular
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Clonagem de Organismos
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Endonucleases
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Sistemas CRISPR-Cas
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Edição de Genes
Limite:
Animals
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Female
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Humans
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Male
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Pregnancy
Idioma:
En
Revista:
Cell Mol Life Sci
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
China