CRISPR and Stem Cells — Changing Cellular Identity and Saving Lives

These cardiomyocytes were reprogrammed from normal adult human skin samples. Credit: Matt Spindler/Gladstone Institutes

Cellular Reprogramming: “The Yamanaka Experiment”

These iPSC-derived neurons were generated from tissue taken from human patients with Huntington’s Disease. The hope for the future is that abnormal cells from patients with inherited diseases such as Huntington’s could be reprogrammed to the pluripotent stem cell state, “edited” using CRISPR and then transplanted into patients to restore normal function. Credit: Julia Kaye/Gladstone Institutes

Far beyond iPSC-based cell replacement therapies

Pictured is a microscopic view of brain cells generated from induced pluripotent stem cells in the laboratory of University of Wisconsin-Madison neuroscientist Su-Chun Zhang. The induced cells, derived from reprogrammed skin cells, seem to have many of the all-purpose qualities of human embryonic stem cells.

iPSC is awesome, but it’s not perfect… yet

Illustration of Single nucleotide polymorphisms (SNP) from DNA Encyclopedia
FMR1 transcription is silenced due to DNA hypermethylation, and the absence of FMRP results in fragile X syndrome. (Retrieved from “Mouse models of the fragile X premutation and fragile X-associated tremor/ataxia syndrome” in Journal of Neurodevelopmental Disorders (6)1:25)
DNA methylation is a process in which methyl (CH3-) groups are added to the DNA molecule. When located in a gene promoter, DNA methylation typically acts to repress gene transcription. (Image from Wikipedia)
Possible methylation and demethylation pathways (Image from Wikipedia)

CRISPR-Cas: Stepping in to Up the iPSC Game

  1. Establish a control group and a test group
  2. Knock out each risk factor gene one by one to see if there is any loss of function
Wild-type human epithelial cells are subjected to gene targeting to create isogenic derivatives that contain a single PIK3CA oncogenic mutation (knock-in) or the same PIK3CA mutation along with a KRAS oncogenic mutation (double knock-in). Cells are then subjected to drugs in parallel to assess resistance sensitivity (J Clin Invest. 2010;120(8):2655–2658. doi:10.1172/JCI44026.)
  • When dCas9 is fused to Kruppel associated box (KRAB), a transcriptional repressor domain, transcription is repressed and the system is referred to as CRISPR interference (CRISPRi).
  • When dCas9 is fused to the SunTag, a sequence containing multiple copies of the activator recruitment domain of general control protein (GCN4), the system activates transcription and is referred to as CRISPR activation (CRISPRa).

… So what do we need to know?

  • We need a replacement for organs in donation, and induced pluripotent stem cells (iPSC) is a promising solution. It has the potential for both replacement therapies and serve as a game-changing model for studying diseases.
  • CRISPR helps establish isogenic cell lines to study the impact of multiple risk factors in polygenic diseases, mainly by generating knock-outs. It also eliminate data noises by turning off single nucleotide polymorphisms (SNP). It gives testing cells a kind of uniformity, so we can find the most effective, reliable iPSC for clinical application.
  • dCas9 activate and repress genes, allowing us to studying their functions. This can be used in genome-wide screenings, one popular type of which is loss-of-function screening.

My Personal Note and Further Reading

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