期刊
PLANT BIOTECHNOLOGY JOURNAL
卷 20, 期 2, 页码 399-408出版社
WILEY
DOI: 10.1111/pbi.13723
关键词
anti-CRISPR; Cas9; anti-CRISPR; Cas12a; AcrIIA4; AcrVA1; gene expression regulation; Nicotiana benthamiana
资金
- Spanish Ministerio de Ciencia e Innovacion, through the Agencia Estatal de Investigacion [PID2019-108203RB-100, PID2020-114691RB-I00, BIO2017-83184-R]
- FPI-UPV fellowship from Universitat Politecnica de Valencia [PAID-01-20]
- European Regional Development Fund
- Generalitat Valenciana [APOSTD/2020/096]
- Fondo Social Europeo post-doctoral grant [APOSTD/2020/096]
- FPI [BIO2016-78601-R]
- Ministerio de Ciencia e Innovacion (Spain) [BIO2016-78601-R, 760331, FPU17/05503]
This study demonstrates that anti-CRISPR proteins AcrIIA4 and AcrVA1 can prevent CRISPR/Cas-mediated gene editing in plants, opening up new possibilities for customized control of gene editing and gene expression.
CRISPR/Cas has revolutionized genome engineering in plants. However, the use of anti-CRISPR proteins as tools to prevent CRISPR/Cas-mediated gene editing and gene activation in plants has not been explored yet. This study describes the characterization of two anti-CRISPR proteins, AcrIIA4 and AcrVA1, in Nicotiana benthamiana. Our results demonstrate that AcrIIA4 prevents site-directed mutagenesis in leaves when transiently co-expressed with CRISPR/Cas9. In a similar way, AcrVA1 is able to prevent CRISPR/Cas12a-mediated gene editing. Moreover, using a N. benthamiana line constitutively expressing Cas9, we show that the viral delivery of AcrIIA4 using Tobacco etch virus is able to completely abolish the high editing levels obtained when the guide RNA is delivered with a virus, in this case Potato virus X. We also show that AcrIIA4 and AcrVA1 repress CRISPR/dCas-based transcriptional activation of reporter genes. In the case of AcrIIA4, this repression occurs in a highly efficient, dose-dependent manner. Furthermore, the fusion of an auxin degron to AcrIIA4 results in auxin-regulated activation of a downstream reporter gene. The strong anti-Cas activity of AcrIIA4 and AcrVA1 reported here opens new possibilities for customized control of gene editing and gene expression in plants.
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