CRISPR-Act3.0 for highly efficient multiplexed gene activation in plants.
Nat Plants
; 7(7): 942-953, 2021 07.
Article
em En
| MEDLINE
| ID: mdl-34168320
ABSTRACT
RNA-guided CRISPR activation (CRISPRa) systems have been developed in plants. However, the simultaneous activation of multiple genes remains challenging. Here, we develop a highly robust CRISPRa system working in rice, Arabidopsis and tomato, CRISPR-Act3.0, through systematically exploring different effector recruitment strategies and various transcription activators based on deactivated Streptococcus pyogenes Cas9 (dSpCas9). The CRISPR-Act3.0 system results in fourfold to sixfold higher activation than the state-of-the-art CRISPRa systems. We further develop a tRNA-gR2.0 (single guide RNA 2.0) expression system enabling CRISPR-Act3.0-based robust activation of up to seven genes for metabolic engineering in rice. In addition, CRISPR-Act3.0 allows the simultaneous modification of multiple traits in Arabidopsis, which are stably transmitted to the T3 generations. On the basis of CRISPR-Act3.0, we elucidate guide RNA targeting rules for effective transcriptional activation. To target T-rich protospacer adjacent motifs (PAMs), we transfer this activation strategy to CRISPR-dCas12b and further improve the dAaCas12b-based CRISPRa system. Moreover, we develop a potent near-PAM-less CRISPR-Act3.0 system on the basis of the SpRY dCas9 variant, which outperforms the dCas9-NG system in both activation potency and targeting scope. Altogether, our study has substantially improved the CRISPRa technology in plants and provided plant researchers a powerful toolbox for efficient gene activation in foundational and translational research.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oryza
/
Engenharia Genética
/
Ativação Transcricional
/
Arabidopsis
/
Solanum lycopersicum
/
Sistemas CRISPR-Cas
/
Melhoramento Vegetal
Idioma:
En
Revista:
Nat Plants
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Estados Unidos