GenoCellEdit, a CRISPR-cas platform for genome editing

Scientific head

Technical participants

Malika Gantier
Research Assistant
Laurent Tesson
Research Assistant
Laure-Hélène Ouisse
Research Assistant

The technology

Gene-specific nucleases currently comprise mainly meganucleases, ZFNs, TALE nucleases and CRISPR-cas 9.  These nucleases recognize and generate DNA double strand cuts by different mechanisms but the cellular DNA repairing machinery will always repair the DNA break by two mechanisms; non-homologous end joining (NHEJ) or homologous recombination if a donor repairing DNA template is provided. NHEJ is an error prone mechanism that generates nucleotide deletions and insertions with shifts of the gene coding frame and gene knockouts. Homologous recombination allows introducing new sequences within the targeted gene. Thus, these gene-specific nucleases allow generating both gene knockouts and knock-ins.

The specific activity of Cas9 endonuclease is easily programmed with a small guide RNA (sgRNA). This simple reprogrammation design has democratized approaches targeted genome modification. Because the overall project is not just the simple design of sgRNA, GenoCellEdit platform provides a complete solution, from consulting in the design of the overall project to the vectorization of CRISPR-Cas system and as well as technical training.

Some applications

  • Gene invalidation (Knock-out)
  • Introduction of an exogenous DNA sequence into a specific locus (Knock-in)
  • Introduction of a specific mutation
  • Genome modification in various biological system (ex: plants, mammalian) and organisms (Ex: human cells, mouse, rat, rice)
  • Gene marking (knock-in of a fluorescent reporter gene)
  • Genetic engineering of induced pluripotent stem cells
  • Resistance to infectious agents
  • Gene therapy: targeted vector integration, gene correction

Platform description

GenoCelEdit is located within the UMR 1064-ITUN, which has a recognized expertise in genome editing and in gene transfer. It aims at promoting genome-editing technology, in particular using CRISPR-cas system. In June 2014, it became open to all academic researchers (in priority) and researchers from private companies. Its R&D activities will support innovative projects to advance the technology and its applications to the common interest.

Know-how

  • in silico sgRNA design>in cellulo functional validation of sgRNA>Design of DNA donor for specific cell genome modification and its detection
  • sgRNA and Cas 9 vectorization (plasmids, RNA, lentiviral vectors, adenoviral vectors)
  • Production of mRNA>Advice on strategy, type of Cas9, vectors and their use

Services

  • Basic: design et validation of sgRNA, Cas 9 and sgRNA vectorization in a plasmid, advices and project support
  • Analyses of sgRNA off-target (off-target) (depending of the species)
  • Design and construction of donor DNA for knock-in 
  • Vectorization as synthetic RNA or in all-in-one viral vector (adenovirus, lentivirus)
  • Technical training for detection of targeted induced mutations (TE7I mutation detection assay)
  • Customizable service:  according to the wishes of the user, some tasks can be shared with the platform for optimal use of respective resources
  • R&D

This activity service is associated to the TRIP plateform (head I. Anegon) and the iPS platform (head L. David, SFR Bonamy) for rat trangenesis and genome engineering of iPS cells, respectively.