Knock-in experiments with LentiFlash®

The delivery of CRISPR/Cas9 system using LentiFlash® particles allows efficient gene knock-in experiments in primary cells while preserving viability and the original cell phenotype.

Gene knock-in with CRISPR/Cas9

  • Beyond gene disruption, the CRISPR technology also allows gene knock-in at a precise genomic site through homology-directed repair (HDR), overcoming safety limitations due to random gene insertion with classical lentiviral vectors.
  • The healthy gene, provided as a DNA donor template, can hence be integrated at its wild-type locus, benefiting from endogenous expression regulation. CRISPR/Cas9 mediated homologous recombination also allows the precise correction of a point mutation. As a result, this mechanism holds great promise for safe and efficient gene therapy.(1)

The delivery of CRISPR/Cas9 system with LentiFlash® for knock-in applications enables to combine the benefits of HDR precision to the fast and transient expression and to the preservation of viability and cell phenotype provided by the LentiFlash particle.

Proof of concept: knock-in using LentiFlash carrying CRISPR-Cas9 in immortalized cells:

  • Monoclonal HCT116-GFP cells (2 copies of GFP/cell) were transduced by LentiFlash® (“LF KO-GFP” expressing Cas9 and a sgRNA targeting GFP) and by an integration-deficient lentiviral vector (IDLV) providing the mCherry DNA donor.
  • HCT116-GFP cells transduced by LF KO-GFP LentiFlash alone were used as a control of GFP editing.
  • Expression of GFP and mCherry was analyzed by FACS, 14 days after transduction, in transduced and non-transduced (NT) cells.

Figure 1: Efficient knock-in of mCherry reporter in monoclonal HCT116-GFP cells
(data gathered by Vectalys)

As shown on figure 1: 8.8% of transduced HCT116-GFP cells expressed mCherry, demonstrating an efficient knock-in of the DNA donor in more than 30% of GFP edited cells.

Knock-in using LentiFlash® carrying CRISPR-Cas9 in primary T cells:

  • T cells from PBMC were activated by CD3/CD28 for 72h, then maintained in culture for 4 days before being transduced by LentiFlash® (“LF.KO PD1” expressing Cas9 and a sgRNA targeting the human PD1 gene) and an integration-deficient lentiviral vector (IDLV), providing the ZsGreen DNA Donor.
  • T cells transduced by LF.KO PD1 LentiFlash alone were used as a control of PD1 editing.
  • 3 days post-transduction, cells were re-activated by CD3/CD28 for 72h.
  • PD1 and ZsGreen expression were analyzed by FACS, 2 days after the second activation, as well as viability and expression of CD3, in transduced and non-transduced (NT) cells.

Figure 2: Efficient knock-in of ZsGreen reporter in primary T cells
(data gathered by Vectalys)

As shown on figure 2: 3.1% of transduced human T cells expressed ZsGreen, demonstrating an efficient knock-in of the DNA donor in more than 5.9% of PD1 edited cells.

Figure 3: Preservation of cell viability and phenotype
(data gathered by Vectalys)

By using highly purified and concentrated LentiFlash particles, human primary T lymphocytes are efficiently transduced without affecting viability. Moreover, TCR expression, evaluated through CD3 quantification, is not impacted by transduction, showing that the original cell phenotype is preserved.

The data above, gathered by Vectalys and shown during the ASGCT 2018, demonstrate that LentiFlash carrying the CRISPR-Cas9 technology can be applied to gene addition through HDR.

Any question about your project ? Do not hesitate to contact us at tech@vectalys.com.

Publications:
1. Maeder, M. L. & Gersbach, C. A. Genome-editing Technologies for Gene and Cell Therapy. Mol. Ther. 24, 430–446 (2016).