Gene Editing Facility

The MCRI Gene Editing Facility will use recently developed technologies to generate gene-edited iPSC lines in feeder-free and chemically defined medium in a manner that is both rapid and cost-effective. A unique feature of our core is the implantation of a one-step gene-editing/reprogramming protocol (Howden et al, 2015) that utilises episomal reprogramming vectors and the Cas9/CRISPR gene-editing system to facilitate the rapid and efficient generation of clonally derived gene-edited iPSC lines from human fibroblasts in the absence of drug selection.

This method is ideal for not only the generation of both genetically repaired iPSCs and their matched isogenic counterparts from patient skin biopsies, but also the rapid generation of knock-in iPSC lines using commercially available (healthy) fibroblasts. We also have capacity to perform gene-editing in already existing human pluripotent stem cell lines and to utilise Cas9 variants that are associated with lower on-target (Howden et at, 2016) and off-target effects (Slaymaker et al., 2016) compared with native Cas9.

Construction/Testing CRISPR vectors
This service includes consultation with the investigator to discuss optimal CRISPR design and targeting strategy. Plasmid DNA containing sgRNA specific to target site of interest will be constructed, purified and sequence verified. The sgRNA plasmid can be validated for nuclease efficiency using a genomic cleavage assay following co-transfection of the CRISPR sgRNA plasmid and Cas9 mRNA into hPSCs. Generation of homologous templates for gene-targeting experiments will also be available upon request. User will be provided with purified plasmid DNA, plasmid map and sequence data.

Cas9 mRNA
mRNA encoding either native Cas9 protein or the NHEJ-deficient version (Cas9-Gem) is available for purchase. Samples will be provided with appropriate QC documentation (spec analysis, gel analysis, flow cytometry analysis demonstrating targeting efficiency in hPSCs).

CRISPR/Cas9-mediated gene knock-out

Service includes:

  • Consultation and generation/validation of sgRNA plasmid(s)
  • Electroporation of sgRNA and Cas9 mRNA into nominated fibroblast or hPSC line
  • PCR screening to identify successfully modified clones
  • DNA Sanger sequencing to verify mutant clones
  • Expansion of up to 3 mutant clones
  • Freezing (4 vials per line)
  • SNP array to assess genomic integrity of at least 2 lines

*Upon request, extra lines can be assessed by SNP array (@ $335 per line + 10% GST for external clients)

At completion (approximately 3 months) user will be provided with:

sgRNA plasmid maps, reports with PCR analysis and Sanger sequencing files confirming successfully modified lines, SNP analyses, mycoplasma free certification. ​

CRISPR/Cas9-mediated gene knock-in or gene repair

Service includes:

  • Consultation and generation/validation of sgRNA plasmid(s)
  • Mycoplasma testing (if cells are provided)
  • Electroporation of sgRNA, Cas9 mRNA and homologous template into nominated fibroblast or hPSC line
  • PCR screening to identify successfully modified clones
  • DNA Sanger sequencing to verify mutant clones
  • Expansion of up to 3 successfully modified clones. Additional matched isogenic controls will also be provided in the case of gene repair experiments.
  • Freezing (4 vials per line)
  • SNP array to assess genomic of at least 2 lines

*Upon request, extra lines can be assessed by SNP array (@ $335 per line + 10% GST for external clients)

At completion (approximately 4 months) user will be provided with:

sgRNA plasmid maps, reports with PCR analysis, Sanger sequencing files confirming successfully modified lines and SNP analyses. 

Disclaimers
If the investigator supplies fibroblasts they can be provided as frozen vials or as live cultures. Cells must test negative for mycoplasma (documentation required) prior to commencement of reprogramming/gene-editing experiments.  Fibroblasts should be at a low passage (ideally <10) and should be actively proliferating.  Senescent cultures are not amenable to reprogramming. In some situations we may not obtain successfully modified lines from the first round. In this scenario a second round will be attempted using a modified strategy (e.g. second sgRNA and/or different starting cell line). The investigator will be charged 30% for two failed attempts.
 

Pricing

Service/ProductInternal ClientsExternal Clients (inclusive of 10% GST)
Generation and validation of CRISPR vectors (sgRNA and donor templates)
$1000 ea​​
​$1100 ea
Cas9 mRNA (50 ug)
$400
$440
Simultaneous reprogramming and gene knock-out of fibroblast line
$5000
$5500
Simultaneous reprogramming and point mutation repair/introduction of fibroblast line
$12,000
​$13,200
Gene knock-out in already existing hPSCs
$7000
$7700
Point mutation repair/introduction in already existing hPSCs
$14,000
$15,400
Reporter line
$14,000
$15,400

​Contact
Sara Howden & Alison Graham
Email: gene.editing@mcri.edu.au​
Phone: (03) 9936 6456​