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CRISPR-Cas9-directed gene tagging using a single integrase-defective lentiviral vector carrying a transposase-based Cas9 off switch.

ABSTRACT: Locus-directed DNA cleavage induced by the CRISPR-Cas9 system triggers DNA repair mechanisms allowing gene repair or targeted insertion of foreign DNA. For gene insertion to be successful, availability of a homologous donor template needs to be timed with cleavage of the DNA by the Cas9 endonuclease guided by a target-specific single guide RNA (sgRNA). We present a novel approach for targeted gene insertion based on a single integrase-defective lentiviral vector (IDLV) carrying a Cas9 off switch. Gene insertion using this approach benefits from transposon-based stable Cas9 expression, which is switched off by excision-only transposase protein co-delivered in IDLV particles carrying a combined sgRNA/donor vector. This one-vector approach supports potent (up to >80%) knockin of a full-length EGFP gene sequence. This traceless cell engineering method benefits from high stable levels of Cas9, timed intracellular availability of the molecular tools, and a built-in feature to turn off Cas9 expression after DNA cleavage. The simple technique is based on transduction with a single IDLV, which holds the capacity to transfer larger donor templates, allowing robust gene knockin or tagging of genes in a single step.

SUBMITTER: Thomsen EA

PROVIDER: S-EPMC9403905 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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CRISPR-Cas9-directed gene tagging using a single integrase-defective lentiviral vector carrying a transposase-based Cas9 off switch.

Thomsen Emil Aagaard EA Skipper Kristian Alsbjerg KA Andersen Sofie S Haslund Didde D Skov Thomas Wisbech TW Mikkelsen Jacob Giehm JG

Molecular therapy. Nucleic acids 20220804

Locus-directed DNA cleavage induced by the CRISPR-Cas9 system triggers DNA repair mechanisms allowing gene repair or targeted insertion of foreign DNA. For gene insertion to be successful, availability of a homologous donor template needs to be timed with cleavage of the DNA by the Cas9 endonuclease guided by a target-specific single guide RNA (sgRNA). We present a novel approach for targeted gene insertion based on a single integrase-defective lentiviral vector (IDLV) carrying a Cas9 off switch ...[more]

PMID: 36090759

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Project description:Viral vectors represent an attractive technology for vaccine delivery. We exploited the integrase defective lentiviral vector (IDLV) as a platform for delivering relevant antigens within the context of the ADITEC collaborative research program. In particular, Influenza virus hemagglutinin (HA) and nucleoprotein (NP) were delivered by IDLVs while H1N1 A/California/7/2009 subunit vaccine (HAp) with or without adjuvant was used to compare the immune response in a murine model of immunization. In order to maximize the antibody response against HA, both IDLVs were also pseudotyped with HA (IDLV-HA/HA and IDLV-NP/HA, respectively). Groups of CB6F1 mice were immunized intramuscularly with a single dose of IDLV-NP/HA, IDLV-HA/HA, HAp alone, or with HAp together with the systemic adjuvant MF59. Six months after the vaccine prime all groups were boosted with HAp alone. Cellular and antibody responses to influenza antigens were measured at different time points after the immunizations. Mice immunized with HA-pseudotyped IDLVs showed similar levels of anti-H1N1 IgG over time, evaluated by ELISA, which were comparable to those induced by HAp + MF59 vaccination, but significantly higher than those induced by HAp alone. The boost with HAp alone induced an increase of antibodies in all groups, and the responses were maintained at higher levels up to 18 weeks post-boost. The antibody response was functional and persistent overtime, capable of neutralizing virus infectivity, as evaluated by hemagglutination inhibition and microneutralization assays. Moreover, since neuraminidase (NA)-expressing plasmid was included during IDLV preparation, immunization with IDLV-NP/HA and IDLV-HA/HA also induced functional anti-NA antibodies, evaluated by enzyme-linked lectin assay. IFNγ-ELISPOT showed evidence of HA-specific response in IDLV-HA/HA immunized animals and persistent NP-specific CD8+ T cell response in IDLV-NP/HA immunized mice. Taken together our results indicate that IDLV can be harnessed for producing a vaccine able to induce a comprehensive immune response, including functional antibodies directed toward HA and NA proteins present on the vector particles in addition to a functional T cell response directed to the protein transcribed from the vector.

Dataset Information

CRISPR-Cas9-directed gene tagging using a single integrase-defective lentiviral vector carrying a transposase-based Cas9 off switch.

Publications

CRISPR-Cas9-directed gene tagging using a single integrase-defective lentiviral vector carrying a transposase-based Cas9 off switch.

Similar Datasets

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