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

SUBMITTER: Thomsen EA 

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

REPOSITORIES: biostudies-literature