title: Lentivirus pre-packed with Cas9 protein for safer gene editing.
abstract: The CRISPR/Cas9 system provides an easy way to edit specific site/s in the genome and thus offers tremendous opportunity for human gene therapy for a wide range of diseases. However, one major concern is off-target effects, particularly with long-term expression of Cas9 nuclease when traditional expression methods such as via plasmid/viral vectors are used. To overcome this limitation, we pre-packaged Cas9 protein (Cas9P LV) in lentiviral particles for transient exposure and showed its effectiveness for gene disruption in cells, including primary T cells expressing specific single guide RNAs (sgRNAs). We then constructed an 'all in one virus' to express sgRNAs in association with pre-packaged Cas9 protein (sgRNA/Cas9P LV). We successfully edited CCR5 in TZM-bl cells by this approach. Using an sgRNA-targeting HIV long terminal repeat, we also were able to disrupt HIV provirus in the J-LAT model of viral latency. Moreover, we also found that pre-packaging Cas9 protein in LV particle reduced off-target editing of chromosome 4:-29134166 locus by CCR5 sgRNA, compared with continued expression from the vector. These results show that sgRNA/Cas9P LV can be used as a safer approach for human gene therapy applications.
authors.affiliation: Department of Biomedical Sciences, Center of Emphasis in Infectious Disease, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, USA.
authors.forename: J G
authors.initials: JG
authors.lastname: Choi
authors.affiliation: Department of Biomedical Sciences, Center of Emphasis in Infectious Disease, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Dang
authors.affiliation: Department of Biomedical Sciences, Center of Emphasis in Infectious Disease, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, USA.
authors.forename: S
authors.initials: S
authors.lastname: Abraham
authors.affiliation: Department of Biomedical Sciences, Center of Emphasis in Infectious Disease, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, USA.
authors.forename: H
authors.initials: H
authors.lastname: Ma
authors.affiliation: Department of Biomedical Sciences, Center of Emphasis in Infectious Disease, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, USA.
authors.forename: J
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Biomedical Sciences, Center of Emphasis in Infectious Disease, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, USA.
authors.forename: H
authors.initials: H
authors.lastname: Guo
authors.affiliation: Department of Biomedicine, Aarhus University, Aarhus, Denmark.
authors.forename: Y
authors.initials: Y
authors.lastname: Cai
authors.affiliation: Department of Biomedicine, Aarhus University, Aarhus, Denmark.
authors.forename: J G
authors.initials: JG
authors.lastname: Mikkelsen
authors.affiliation: Department of Biomedical Sciences, Center of Emphasis in Infectious Disease, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, USA.
authors.forename: H
authors.initials: H
authors.lastname: Wu
authors.affiliation: Department of Biomedical Sciences, Center of Emphasis in Infectious Disease, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, USA.
authors.forename: P
authors.initials: P
authors.lastname: Shankar
authors.affiliation: Department of Biomedical Sciences, Center of Emphasis in Infectious Disease, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, USA.
authors.forename: N
authors.initials: N
authors.lastname: Manjunath
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D002894:Chromosomes, Human, Pair 4 / Q000235:genetics; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006678:HIV / Q000235:genetics; D006801:Humans; D019169:Jurkat Cells; D016086:Lentivirus / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D019713:Receptors, CCR5 / Q000235:genetics / Q000378:metabolism; D020079:Terminal Repeat Sequences; D019065:Virus Assembly
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D019713:Receptors, CCR5; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases