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January 20, 2021

In a previous post we provided an overview of induced pluripotent stem cells (iPSCs), and some considerations for how they can be used in cell and gene therapies (CGTs). Here we will look at genetic engineering approaches for iPSCs and explore technical challenges and solutions.

How can iPSCs be Genetically Engineered?

iPSCs are typically modified in two main ways:

  1. Knock-out mutation to inactivate a gene  
  2. Knock-in mutation to add/modify a gene sequence      

An important focus in the field has been generating iPSC lines that model human disease. This can be accomplished in a few different ways.

One approach is to generate patient-specific iPSC lines from individuals with a particular disease. CCRM has extensive experience with this approach and has been collaborating with Toronto researcher Christine Bear to create patient-specific iPSC lines from individuals with cystic fibrosis (CF). Gene editing approaches are applied to correct the disease-causing mutations and learn more about the molecular mechanisms responsible for CF.

Our team also has the capability to use gene editing to introduce disease causing mutations (e.g. single nucleotide polymorphisms (SNPs)) into normal iPSC lines to model different diseases.

Creating Process Improvements that Make Editing More Efficient

iPSCs are not easily amenable to genetic manipulation. We have overcome this challenge by developing a robust process for generating gene-modified iPSCs. We rely on well-developed protocols that effectively introduce gene edits and speed-up the process for identifying populations of iPSCs that carry the genetic modification of interest. We have optimized a nucleofection-based protocol for introducing the CRISPR gene editing system into iPSCs.

The strength of our editing platform is a digital droplet PCR (ddPCR)-based screening approach that allows for fast and accurate identification of single cell clones that have the desired edits. ddPCR screening is confirmed by DNA sequencing and pluripotency markers are tested using flow cytometry and qPCR.

Solutions to Common Challenges for Gene Editing iPSCs

Generating and testing a gene-edited iPSC line takes six months on average. This process is technically challenging and requires specialized expertise. It is important to remember that not all gene edits are created equal. The properties of the genetic locus where the edit is targeted may restrict editing ability and make editing less efficient. The gene editing process is stressful for iPSCs and can cause unwanted outcomes, such as spontaneous differentiation or karyotype changes. Therefore, testing for karyotype abnormalities is an important quality control step for edited iPSCs.

To further ensure the safety of edited iPSCs that are generated using CRISPR, it is important to screen for off-target gene edits.

Finally, determining the clonality of iPSCs is critical to ensuring a homogenous edited cell line. Comparison of short tandem repeat (STR) DNA sequences is used to confirm that edited iPSCs are derived from the same parental cells.

In summary, our experts can help you overcome technical challenges by applying specialized skills and experience to the design of custom solutions for your gene-edited iPSC line. Contact us (cdmo@ccrm.ca) to find out how our team can help with your iPSC editing needs.

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