Integrase-Deficient Lentiviral Vector as an All-in-One Platform for Highly Efficient CRISPR/Cas9-Mediated Gene Editing

The CRISPR/Cas9 systems have revolutionized the field of genome editing by providing unprecedented control over gene sequences and gene expression in many species, including humans. Lentiviral vectors (LVs) are an important means of delivering CRISPR/Cas9 components due to their ability to accommodate large DNA payloads and efficiently transduce a wide range of dividing and non-dividing cells. However, permanently expressed CRISPR/Cas9 may facilitate undesirable off-target effects, hindering their utility for genome-editing applications that require high levels of precision. Episomal integrase-deficient lentiviral vectors (IDLVs) are an ideal platform for efficient delivery of large genetic cargos where only transient expression of the transgene is desired.

In this manuscript, the scientists aimed to establish IDLVs as a means for safe and efficient delivery of CRISPR/Cas9. To this end, they developed an all-in-one vector cassette with increased production efficacy and demonstrated that CRISPR/Cas9 delivered by the improved IDLV vectors can mediate rapid and robust gene editing in several experimental contexts.

First, they compared IDLV and integrating LV all-in-one to knockout GFP stably expressed in HEK293T. Their data show ∼5-fold reduction in the number of GFP-positive cells as early as 7 days pt, with a nearly complete signal depletion observed by 21 days pt with both integrated and episomal vectors. These results clearly demonstrate that CRISPR/Cas9 delivered by IDLVs is capable of mediating rapid, robust, and sustained gene editing in dividing cells. 

Then, they tested the IDLV-sgRNA/Cas9 platform as an in vivo gene editing system by targeting expression of a γ-amino-butyric acid A (GABAA) receptor subunit α2 in the nucleus accumbens (NAc) of adult male Sprague-Dawley rats using an IDLV-α2/Cas9 vector. At 35–47 days following microinjection of the IDLV α2/Cas9 vector, α2 protein declined to undetectable levels. These findings highlight the utility of IDLV sgRNA/Cas9 platform for long-term reduction of gene function in non-dividing brain cells.

IDLVs present an attractive platform for viral-mediated gene transfer due to their numerous advantages over other delivery methods: (1) they are capable of efficiently transducing a broad range of cells and tissues, (2) they have large packaging capacity, (3) they demonstrate low cytotoxicity and immunogenicity, and (4) they retain very weak integration capacity and are transiently expressed from episomal genomes. These advantages make IDLVs a powerful tool in basic science and clinical research.

Furthermore, it is very easy now to obtain ultra-high performant IDLV fully customized thanks to Biotech such as GEG Tech which provides two-vector system and all-in-one system with IDLV or integrating LVs platforms. Moreover, last generation of LV developed by GEG Tech is deficient for reverse transcriptase and remains into RNA phase. This new LV generation upgrades the level of LVs safety while keeping the advantages of this gene transfer tool.

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