Subculturing cells have no effect on CRISPR/Cas9‐mediated cleavage of UL30 gene in pseudorabies virus

Abstract

CRISPR /Cas9‐mediated genome editing can inhibit virus infection by targeting the conserved regions of the viral genomic DNA . Unexpectedly, we found previously that pseudorabies virus ( PRV ) could escape from CRISPR /Cas9‐mediated inhibition. In order to elucidate whether the escape of PRV from Cas9‐mediated inhibition was due to cell deficiencies, such as genetic instability of sg RNA or Cas9 protein, the positive cells were passaged ten times, and PRV infection in the sg RNA ‐expressing cells was evaluated in the present study. The results showed that subculturing cells has no effect on Cas9‐mediated cleavage of PRV . Different passages of PX 459‐ PRV cells can stably express sg RNA to facilitate Cas9/sg RNA cleavage on the UL 30 gene of PRV , resulting in a pronounced inhibition of PRV infection. Studies to elucidate the mechanism of PRV escape are currently in progress.

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“…In another study, 75 sgRNAs targeting different genes across the PRV genome were designed and transfected into the Vero cell line, a significant decline of PRV infection and replication was observed with most of the designed sgRNAs (Tang et al 2017). A significant decrease in virus replication and yield in each passage was observed in PRV infected cell line (PX459) which stably expressed Cas9 nuclease and specific sgRNA targeting UL30 gene which is conserved in PRV (Ren et al 2018). This indicates that it is the efficient editing of CRISPR/Cas9 which has led to a significant decline of viral replication.…”

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“…In 2015, some researchers co-transfected the purified PRV genomes with the constructed specific gRNA CRISPR/Cas9 complex into PK15 cells and obtained up to 100% viral gene editing efficiency [ 77 ]. Simultaneously, a cell line stably expressing Cas9 nuclease and sgRNA targeting the UL30 gene conserved in PRV was developed, finding that the UL30 gene of infected PRV can be cleaved efficiently, and the stable expression of Cas9 nuclease has no adverse effect on the proliferation of PRV [ 78 ]. Compared to the single gRNA-based transfection-infection approach, the double-gRNA strategy demonstrated a significantly better knockout or knockin efficiency for manipulating PRV viral genes [ 79 ].…”