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Strong and tunable anti-CRISPR/Cas activities in plants
Plant Biotechnology Journal ( IF 13.8 ) Pub Date : 2021-10-10 , DOI: 10.1111/pbi.13723
Camilo Calvache 1 , Marta Vazquez-Vilar 1 , Sara Selma 1 , Mireia Uranga 1 , Asun Fernández-Del-Carmen 1 , José-Antonio Daròs 1 , Diego Orzáez 1
Affiliation  

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.

中文翻译:

植物中强大且可调节的抗 CRISPR/Cas 活性

CRISPR/Cas 彻底改变了植物的基因组工程。然而,尚未探索使用抗 CRISPR 蛋白作为防止植物中 CRISPR/Cas 介导的基因编辑和基因激活的工具。本研究描述了本氏烟草中两种抗 CRISPR 蛋白 AcrIIA4 和 AcrVA1 的特性。我们的研究结果表明,当与 CRISPR/Cas9 瞬时共表达时,AcrIIA4 可防止叶片中的定点诱变。以类似的方式,AcrVA1 能够阻止 CRISPR/Cas12a 介导的基因编辑。此外,使用组成性表达 Cas9 的本氏烟草线,我们表明使用烟草蚀刻病毒对 AcrIIA4 的病毒传递能够完全消除当引导 RNA 与病毒一起传递时获得的高编辑水平,在这种情况下是马铃薯病毒X。我们还表明,AcrIIA4 和 AcrVA1 抑制基于 CRISPR/dCas 的报告基因转录激活。在 AcrIIA4 的情况下,这种抑制以高效、剂量依赖性的方式发生。此外,生长素degron 与AcrIIA4 的融合导致下游报告基因的生长素调节激活。本文报道的 AcrIIA4 和 AcrVA1 的强抗 Cas 活性为植物基因编辑和基因表达的定制控制开辟了新的可能性。
更新日期:2021-10-10
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