Efficient CRISPR gene editing in primary cells and organoids using virus-like particles

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Scope of the method

The Method relates to
  • Human health
The Method is situated in
  • Basic Research
  • Translational - Applied Research
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Human derived cells / tissues / organs
Specify the type of cells/tissues/organs
several primary cell models (rectal organoids, endothelial cells, epithelial cells, iPSCs, ...)

Description

Method keywords
  • organoids
  • genome engineering
  • gene editing
  • CRISPR-Cas9
  • CRISPR/Cas
  • virus-like particles
  • transduction
  • base edting
  • prime editing
  • knock out
Scientific area keywords
  • gene therapy
  • genetics
  • 3D organoid models
  • human diseases
  • human health
  • human adult stem cells
Method description

Using the combination of advanced CRIPSR tools including several Cas orthologs, based editors (ABE, CBE, CGBE) and prime editing technologie with efficient delivery vehicles such as LV, AAV and virus-like particles (VLPs), our lab has become experienced with introducing or replacing precise edits in several primary cell models. For gene editing, VLPs are especially well suited because they link the efficacy of viral transduction with the delivery of RNP cargo, delivering a very transient dose of gene editing cargo. We have built VLPs harbouring several gene editing enzymes (Cre, several Cas variants, several base editors, several PE strategies), as well as reporter cargos (mNeonGreen, B-galactosidase, fLuc,...).

Lab equipment

BSL2 is required to work with cells from human origin and with particles capable of entering these cells.

Method status
  • Still in development
  • Internally validated

Pros, cons & Future potential

Advantages

Efficient gene editing in hard-to-edit cell types using ultra transient exposure and therefore limiting risks on off-target editing. VLP preps can be ordered from the Leuven Viral Vector Core (https://gbiomed.kuleuven.be/english/corefacilities/LVVC/technology)

Challenges

VLP production is expensive and needs to be performed by experienced staff under highly standardised SOP's. We have large libraries of VLPs available and have shared many before under academic collaboration.

References, associated documents and other information

Links
Leuven Viral Vector Core
Other remarks

This work was performed in collaboration with the Leuven Viral Vector Core (Contact: lvvc@kuleuven.be).

Contact person

Marianne Carlon

Organisations

Katholieke Universiteit Leuven (KUL)
Chronic Diseases and Metabolism
Laboratory of Respiratory Diseases and Thoracic Surgery (BREATHE)
Belgium
Flemish Region

Katholieke Universiteit Leuven (KUL)
Pharmaceutical and Pharmacological Sciences
Belgium
Flemish Region

Partners

Katholieke Universiteit Leuven (KUL)