Journal of Virology: MULTIPLEXED SIV-SPECIFIC PAIRED RNA-GUIDED CAS9 NICKASES INACTIVATE PROVIRAL DNA (Lai)
Authors: Lisa M Smith, Jason T. Ladner, Vida L. Hodara, Laura M. Parodi, R. Alan Harris, Jessica E. Callery, Zhao Lai, Yi Zou, Muthuswamy Raveedran, Jeffrey Rogers, and Luis D. Giavedoni
Human and simian immunodeficiency virus infections establish a lifelong reservoir of cells harboring an integrated proviral genome. Genome editing CRISPR-associated Cas9 nucleases, combined with SIV-specific guiding RNA (gRNA) molecules, inactive integrated provirus DNA in vitro and in animal models. We generated RNA-guided Cas9 nucleases (RGNu) and nickases (RGNi) targeting conserved SIV regions with no homology in the human or rhesus macaque genome. Assays in cells co-transfected with SIV provirus and plasmids coding for RGNus identified SIV LTR, TAR, and RSS regions as the most effective at virus suppression; RGNi targeting these same regions inhibited virus production significantly. Multiplex plasmids that co-expressed these three RGNu (Nu3), or six (three pairs) RGNi (Ni6), were more efficient at virus suppression than any combination of individual RGNu and RGNi plasmids. Both Nu3 and Ni6 plasmids were tested in lymphoid cells chronically infected with SIVmac239, and whole-genome sequencing was used to determine on- and off-target mutations. Treatment with these all-in-one plasmids resulted in similar levels of mutations of viral sequences from the cellular genome; Nu3 induced indels at the 3 SIV-specific sites, whereas for Ni6 indels were present at the LTR and TAR sites. Levels of off-target effects detected by two different algorithms were indistinguishable from background mutations. In summary, we demonstrate that Cas9 nickase in association with gRNA pairs can specifically eliminate parts of the integrated provirus DNA; also, we show that careful design of an all-in-one plasmid coding for 3 gRNAs and Cas9 nuclease inhibits SIV production with undetectable off-target mutations making these tools a desirable prospect for moving into animal studies.
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