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Genome Biology

, 16:238

Genome editing

Abstract

BackgroundThe CRISPR-Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR-Cas9 has been used for targeted genome editing in diverse eukaryotic species.

ResultsIn this study, we investigate whether the CRISPR-Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus TYLCV into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR-Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region IR are the most effective. N. benthamiana plants expressing CRISPR-Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses.

ConclusionsThese data establish the efficacy of the CRISPR-Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.

KeywordsPlant genome engineering CRISPR-Cas9 system Synthetic site-specific nucleases Viral-mediated genome editing Virus resistance Tomato yellow leaf curl virus Tobacco rattle virus AbbreviationsBCTVbeet curly top virus

bpbase pair

CasCRISPR-associated

CPcapsid protein

CRcommon region

CRISPRclustered regularly interspaced palindromic repeat

dpidays post-infiltration

dsDNAdouble-stranded DNA

IRintergenic region

MeMVMerremia mosaic virus

NBN. benthamiana

OEoverexpressing

ORFopen reading frame

PAMprotospacer-associated motif

PCRpolymerase chain reaction

PTGpolycistronic tRNA–gRNA

RCArolling-circle amplification

sgRNAsingle guide RNA

ssDNAsingle-stranded DNA

TRVtobacco rattle virus

TYLCVtomato yellow leaf curl virus

Electronic supplementary materialThe online version of this article doi:10.1186-s13059-015-0799-6 contains supplementary material, which is available to authorized users.

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Autor: Zahir Ali - Aala Abulfaraj - Ali Idris - Shakila Ali - Manal Tashkandi - Magdy M. Mahfouz

Fuente: https://link.springer.com/



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