title: Specific induction of endogenous viral restriction factors using CRISPR/Cas-derived transcriptional activators.
abstract: Whereas several mammalian proteins can restrict the replication of HIV-1 and other viruses, these are often not expressed in relevant target cells. A potential method to inhibit viral replication might therefore be to use synthetic transcription factors to induce restriction factor expression. In particular, mutants of the RNA-guided DNA binding protein Cas9 that have lost their DNA cleavage activity could be used to recruit transcription activation domains to specific promoters. However, initial experiments revealed only weak activation unless multiple promoter-specific single guide RNAs (sgRNAs) were used. Recently, the recruitment of multiple transcription activation domains by a single sgRNA, modified to contain MS2-derived stem loops that recruit fusion proteins consisting of the MS2 coat protein linked to transcription activation domains, was reported to induce otherwise silent cellular genes. Here, we demonstrate that such "synergistic activation mediators" can induce the expression of two restriction factors, APOBEC3G (A3G) and APOBEC3B (A3B), in human cells that normally lack these proteins. We observed modest activation of endogenous A3G or A3B expression using single sgRNAs but high expression when two sgRNAs were used. Whereas the induced A3G and A3B proteins both blocked infection by an HIV-1 variant lacking a functional vif gene by inducing extensive dC-to-dU editing, only the induced A3B protein inhibited wild-type HIV-1. These data demonstrate that Cas9-derived transcriptional activators have the potential to be used for screens for endogenous genes that affect virus replication and raise the possibility that synthetic transcription factors might prove clinically useful if efficient delivery mechanisms could be developed.
authors.affiliation: Department of Molecular Genetics and Microbiology, Center for Virology, Duke University Medical Center, Durham, NC 27710.
authors.forename: Hal P
authors.initials: HP
authors.lastname: Bogerd
authors.affiliation: Department of Molecular Genetics and Microbiology, Center for Virology, Duke University Medical Center, Durham, NC 27710.
authors.forename: Anand V R
authors.initials: AV
authors.lastname: Kornepati
authors.affiliation: Department of Molecular Genetics and Microbiology, Center for Virology, Duke University Medical Center, Durham, NC 27710.
authors.forename: Joy B
authors.initials: JB
authors.lastname: Marshall
authors.affiliation: Department of Molecular Genetics and Microbiology, Center for Virology, Duke University Medical Center, Durham, NC 27710.
authors.forename: Edward M
authors.initials: EM
authors.lastname: Kennedy
authors.affiliation: Department of Molecular Genetics and Microbiology, Center for Virology, Duke University Medical Center, Durham, NC 27710 bryan.cullen@duke.edu.
authors.forename: Bryan R
authors.initials: BR
authors.lastname: Cullen
mesh_terms: D000071480:APOBEC-3G Deaminase; D001483:Base Sequence; D015153:Blotting, Western; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D003564:Cytidine Deaminase / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D015778:Minor Histocompatibility Antigens; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation; D054320:vif Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism
chemical_list: D015778:Minor Histocompatibility Antigens; D054320:vif Gene Products, Human Immunodeficiency Virus; C514847:vif protein, Human immunodeficiency virus 1; D000071480:APOBEC-3G Deaminase; C495794:APOBEC3B protein, human; C464924:APOBEC3G protein, human; D003564:Cytidine Deaminase
keywords: APOBEC3 proteins; CRISPR/Cas; HIV-1; restriction factors; synthetic transcription factors