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One-bp Substitution in the 3 Billion-bp Human Genome

Genome editing allows for any manipulations of the genome in human iPS cells, which can be applied for disease modeling and cell transplantation therapy. However, it had been difficult to substitute only one base-pair (bp) in the 3 billion-bp human genome without leaving artificial DNA sequences. One-bp substitution by genome editing can serve as a versatile platform for various applications, because many diseases are caused by single-bp mutations.

 We developed a method for isolation of iPS cells with only single-bp substitutions by combining CRISPR/Cas9, TALEN, and digital PCR (Fig. 1, Miyaoka Nature Methods 2014).

 By using our method, we can introduce single-bp substitutions at any locations in the genome (Fig. 2).

Fig. 1

Detection of Genome Editing Events by Digital PCR

We use digital PCR to detect genome editing events in cells in a sensitive and quantitative manner. For details, please watch a webinar 

(TheScientist Webinar Series, Oct 14th, 2015).


Fig. 3

Wild-type cell

Mutant cell


Modeling Disease in a Dish to Study Pathogenesis

By editing the genome in iPS cells, we can study pathogenesis of genetic disorders in any cell types in a dish. For example, a point mutation of cardiomyopathy introduced into iPS cells caused abnormal sarcomere structure (a functional unit of muscle contraction visualized as red stripes), when these cells were differentiated into cardiomyocytes (Fig. 3).  We revealed a pathogenic mechanism by abnormal aggregation of RNA and proteins unexpectedly shared between cardiomyopathy and neurodegenerative diseases.

Seeking for Precise Genome Editing Conditions for Therapy 

iPS cells from patients with genetic disorders maintain the causative mutations. However, if we correct these mutations, the patient-derived iPS cells can potentially be used for cell therapy. For this purpose, we need genome editing that does not cause any unwanted damages to the genome. We will correct pathogenic mutations such as ones of Wilson's disease (a genetic liver disorder) in iPS cells with diverse genome editing conditions to evaluate their accuracy by using highly sensitive digital PCR and other techniques.

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