Gene Editing Facility | CRISPR | Cas9

The iPSC Derivation & Gene Editing Core is a facility established and managed by the MCRI Stem Cell Medicine Initiative and has been in operation since 2014. This facility brings together a team of highly experienced stem cell and molecular biologists with over 40 years combined experience to generate standard and 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 implementation of a streamlined one-step gene-editing/reprogramming protocol 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 primary fibroblasts or blood. We also have capacity to perform gene-editing in already existing human pluripotent stem cell lines as well as the generation of lineage-specific reporter lines. 

Upon initial contact, we liaise with customers to devise the ideal strategy for achieving a successful gene-editing outcome based upon the ever-expanding number of gene-editing tools available including: 

• Wildtype spCas9 
• pCas9 variants (Cas9-Gem, Cas9-Cdt1 and HifiCas9) 
• Cas12 
• ABE and CBE base-editing Cas9 variants 
• Primer-editing 

The iPSC Derivation & Gene Editing Core also offers other key services such as teratoma assays, MEF production, and customised training. 

Our Services: 

Generation of patient iPS cell lines 

Service includes: 

• Episomal or Sendai delivery of reprograming factors 
• From fibroblast, peripheral/cord blood cells and PBMCs 
• iPSCs generated can be cultured to the requirements of the end user (on MEFs or MEF-free) 
• iPSCs supplied as QA checked clones (3 clones per cell line; flow cytometry for pluripotency markers, karyotyping via SNP analysis and mycoplasma tested) expanded and cryo-preserved for the user 
• Provision of a standard report 

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, 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 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, user will be provided with: sgRNA plasmid maps, reports with PCR analysis, Sanger sequencing files confirming successfully modified lines and SNP analyses, mycoplasma free certification. 

Blood processing and cryopreservation 

• Isolation of peripheral blood mononuclear cells (PBMCs) from whole blood in EDTA/Heparin/Citrate tubes stored at RT 
• Cryopreservation 
• Short term storage in LN2 when requesting generation of iPSCs 

Culture reagents for the maintenance of iPSC lines 

Preparation and provision of basic medias and mouse embryonic feeder cells (MEFs) 

Teratomas  

• Supply of mice and preparation of cell lines to be injected 
• Subcutaneous injection of undifferentiated cells 
• Subcutaneous injection/implant of differentiated cells also available 
• Monitoring and teratoma detection  
• Teratoma harvest and provision to researcher 

hES/iPS cell culture service  

• Services include but not limited to: 
• Clone derivation  
• Cell maintenance  
• Cell expansion for cryopreservation  

MEF and MEF-free options available 

Cell lines must test negative for mycoplasma (documentation required) prior to entering the facility.  

Training and support  

We offer customised one-on-one training which is complimentary for researchers that use our iPSC derivation and/or gene-editing services. We offer specific training around iPSC maintenance and passaging using various formats (feeder and feeder-free methods) as well as cryopreservation and revival of iPSCs. Ongoing advisory and technical assistance covering all aspects of culture. 

Disclaimers  

If the investigator supplies primary 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. Primary 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. 

For primary fibroblast lines that reprogram poorly using the simultaneous reprogramming/gene editing approach, iPSC lines will be generated first and then subsequently gene edited to obtain enough clones to screen. In these instances, we will charge the equivalent of gene editing an already established iPSC line, i.e., $14,000 ($15,400 including GST). 

Pricing

Price does not include shipment of cell lines on dry ice which can be organised with your choice or courier.

​Contact
Email: ips.gene@mcri.edu.au
Phone: (03) 9936 6456​ OR (03) 9936 6164

Role: 

Team Leader/Director Gene Editing Facility

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