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Abstract
Clustered Regularly Interspaced Short Palindromic Repeat activation-synergistic activation mediator system (CRISPRa-SAM) has been efficiently used to up-regulate the targeted genes in human and mouse. But it is not known whether the CRISPRa-SAM system can be used against porcine disease because its two important transcriptional activation domains (P65 and heat shock transcription factor 1 (HSF1)) are from mouse and human, respectively. Pig is one of the most important meat sources, porcine viral infectious diseases cause massive economic losses to the swine industry and threaten the public health. We aimed to investigate whether the CRISPRa-SAM system could increase porcine antiviral activity by mediating two pig-specific target genes (Mx2 and β1,4 N-acetylgalactosaminyltransferase (B4galnt2)). First, we constructed PK-15 and IPEC-J2 cell lines expressing nuclease-deficient Cas9 (dCas9)-vp64 and MS2-P65-HSF1 stably. Next, in these two cell models, we activated Mx2 and B4galnt2 expression through CRISPRa-SAM system. Antiviral activity to PRV or H9N2 was improved in PK-15 cells where Mx2 or B4galnt2 was activated. Altogether, our results demonstrated the potential of CRISPRa-SAM system as a powerful tool for activating pig genes and improving porcine antiviral activity.
Highlights
Pig is one of the most important meat producing animals
We checked the expression till day 14 post selection, for important components of CRISPRa-SAM system by infecting PK-15 and IPEC-J2 cells with lentiviruses expressing deficient Cas9 (dCas9)-VP64 and MS2-P65-heat shock transcription factor 1 (HSF1), respectively
The CRISPRa-SAM system is capable of activating the target gene with only an sgRNA regardless the size of target gene
Summary
Pig is one of the most important meat producing animals. Porcine viral infectious diseases cause massive economic losses to the swine industry. The Clustered Regularly Interspaced Short Palindromic Repeat activation-synergistic activation mediator system (CRISPRa-SAM) system needs to be explored for pig antiviral breeding. This system is an extension of the conventional CRISPR knockout system. The dCas is fused to herpes simplex virus (HSV) viral protein 16 (VP16) activation domains (known as VP64) and the system includes some synergistic activation mediators. These mediators comprise fusion proteins (MS2-guide RNA (gRNA), dCas9-VP64 and the MS2-P65-HSF1), MS2 bacteriophage coat protein fused to the NF-κB (P65) and heat shock transcription factor 1 (HSF1)
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