Production and Validation of Lentiviral Vectors for CRISPR/Cas9 Delivery.

ABSTRACT

Genetic information transferred by HIV-1-based lentiviral vectors as single-stranded RNA is converted to double-stranded DNA by reverse transcription and subsequently inserted into the genome of recipient cells. Integration into the genome allows stable, long-term expression of genes-of-interest driven by promoter sequences contained within the vector. This technology can be used as a standard method for production of cells stably expressing Cas9 protein and single guide RNA (sgRNA), the key components of the CRISPR genome editing system. Here, we provide a protocol for production and validation of VSV-G-pseudotyped lentiviral vectors for delivery of the CRISPR system and generation of knockout cell lines.