Multiplexed Simian Immunodeficiency Virus-Specific Paired RNA-Guided Cas9 Nickases Inactivate Proviral DNA.
J Virol
; 95(23): e0088221, 2021 11 09.
Article
em En
| MEDLINE
| ID: mdl-34549979
ABSTRACT
Human and simian immunodeficiency virus (HIV and SIV) infections establish lifelong reservoirs of cells harboring an integrated proviral genome. Genome editing CRISPR-associated Cas9 nucleases, combined with SIV-specific guiding RNA (gRNA) molecules, inactivate 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 cotransfected with SIV provirus and plasmids coding for RGNus identified SIV long terminal repeat (LTR), trans-activation response (TAR) element, and ribosome slip site (RSS) regions as the most effective at virus suppression; RGNi targeting these regions inhibited virus production significantly. Multiplex plasmids that coexpressed 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. IMPORTANCE Our approach to HIV cure, utilizing the translatable SIV/rhesus macaque model system, aims at provirus inactivation and its removal with the least possible off-target side effects. We developed single molecules that delivered either three truncated SIV-specific gRNAs along with Cas9 nuclease or three pairs of SIV-specific gRNAs (six individual gRNAs) along with Cas9 nickase to enhance efficacy of on-target mutagenesis. Whole-genome sequencing demonstrated effective SIV sequence mutation and inactivation and the absence of demonstrable off-target mutations. These results open the possibility to employ Cas9 variants that introduce single-strand DNA breaks to eliminate integrated proviral DNA.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
DNA
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Provírus
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Vírus da Imunodeficiência Símia
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RNA Guia de Cinetoplastídeos
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Desoxirribonuclease I
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article