What will spark when iPSC meets CRISPR? | CRISPR-U™-mediated gene-editing iPSC


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Location:Home > Application > What will spark when iPSC meets CRISPR? | CRISPR-U™-mediated gene-editing iPSC

What will spark when iPSC meets CRISPR? | CRISPR-U™-mediated gene-editing iPSC


 Induced pluripotent stem cell (iPSC) is a pluripotent stem cell formed by dedifferentiation of a mammalian somatic cell by some phases such as being transferred with transcription factors. It was first discovered by the research team of Japanese scholar Shinya Yamanaka in 2006. The induction method is to transfer Oct3/4, Sox2, c-Myc, and Klf4 transcription factors into mouse somatic cells through lentivirus to convert them into pluripotent stem cells, which are similar to embryonic stem cells. In 2007, iPSC induction technology was successfully applied to human cells. Since then, researchers have continuously optimized the induction methods, such as plasmid transfection, adenovirus infection, synthetic RNAs and proteins and other methods without exogenous gene integration, to improve the safety of iPSC application.

iPSC and embryonic stem cells (ESC) have similar regenerative ability. Theoretically, iPSC can differentiate into all organs, tissues or cells, which to a large extent solves the problem of difficult access to nerve cells, cardiomyocytes and other cells. Compared with ESC, iPSC has a wide range of sources and is easy to obtain, and does not need to destroy embryos, so it faces fewer ethical disputes. Moreover, iPSC derived from patients themselves can also avoid the risk of immune rejection in cell transplantation. Therefore, iPSC has impacted the place of ESC to a certain extent and are considered to have a wider application prospect in the asepcts of regenerative medicine, new drug development, disease model generation, etc.

However, there is a problem with iPSC as a disease model, that is, it is difficult to distinguish whether the disease phenotype is caused by the mutation of the target gene or by the difference in genetic background and environmental factors of different individuals. In order to overcome this problem, researchers used gene editing technology and iPSC to create an isogenic model (i.e., with the same genetic background) as the control, and it can well exclude the interference factors other than the mutations. The specific method is to carry out mutation repair on iPSCs derived from patients and observe whether the disease phenotype disappears after induction and differentiation, or to mutate iPSCs derived from normal people and observe whether the disease phenotype appears after induction and differentiation. CRISPR/Cas9, as the most mainstream gene-editing technology in recent years, has the advantages of high efficiency, simple operation and low cost. It has been widely used in iPSC-related gene knockout, point mutation and knock-in. Here are some case studies.


Disease modeling: Construction of iPSC model of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease, with an incidence rate of about one in 400-1000 people. About half of patients with this disease will develop end-stage renal disease, requiring dialysis or kidney transplantation. ADPKD is associated with two gene defects, of which 85% are caused by mutations in the gene PKD1 (TRPP1) located on chromosome 16. Romano et al. generated isogenic iPSC with heterozygous knockout and homozygous knockout in PKD1 gene by using CRISPR/Cas9 technology, and verified that iPSC maintained stem cell like morphology, normal karyotype, pluripotency and differentiation ability in three germ layers. It can be used as a model for studying the pathogenesis of ADPKD and drug screening. [1]



Mutation repair: Patient-derived iPSC recovered the expression of β- Globin (HBB) after mutation repaired

β-Thalassemia is a monogenic disease caused by point mutation or fragment deletion of the HBB gene leading to loss of normal β-globin peptide chain or insufficient synthesis of β-globin. The prevalence of β- thalassemia carriers in southern China is 2.54%, which has been threatening the lives of millions of people for decades. At present, hematopoietic stem cell transplantation is the only available radical treatment for patients with severe β-thalassemia. However, hematopoietic stem cell transplantation is limited by the lack of HLA-matched healthy donors in most patients. With the wide application of gene-editing technology, researchers used CRISPR/Cas9 system to perform gene-editing on patient-derived iPSCs (genotype: homozygous 41/42 deletion): the gRNAs targeting HBB mutation position were mainly designed, and the recombination was carried out with the PCR products containing normal sequences (WT) as the donor template, and then the cells with mutation repaired genotype were screened. The successfully corrected iPSCs were re-differentiated into hematopoietic stem cells (HSCs), and then the HSCs were transplanted to the irradiated NSI immunodeficient mice. Finally, it was found that the mice transplanted with the corrected iPSCs could have normal hematopoiesis and produce normal HBB protein. This study brings new hope for the treatment of β-thalassemia. [2]


Antiviral treatment: iPSC and its derived blood cells have HIV resistance after knockout of CCR5

Chemokine receptor 5 (CCR5), as a co-receptor of HIV virus, is essential for the cellular infection of CCR5 tropic virus. Studies have shown that the loss of CCR5 function can prevent HIV infection. Through gene editing of immune cells in patients, it is possible to treat HIV-infected patients. However, due to the low transfection efficiency of immune cells and the difficulty of cell culture, it is difficult to directly genetically edit these cells. Kang et al. performed gene-editing on iPSC by using CRISPR/Cas9 technology, designed a single gRNA knockout strategy and a double gRNA knockout strategy targeting CCR5, and successfully screened the CCR5 knockout homozygous clones. iPSCs with homozygous CCR5 knockout still showed the typical characteristics of pluripotent stem cells and can effectively differentiate into hematopoietic cells and macrophages. In vitro HIV infection experiment showed that the KO cells had unique resistance to the attack of CCR5 tropic virus. This study shows that combining iPSC technology with CRISPR/Cas9 technology has a promising application in the treatment of HIV infection. [3]

In fact, there are many cases of CRISPR gene-editing application in iPSCs for disease model construction and cell therapy, such as neurodegenerative diseases, metabolic diseases and cardiac genetic diseases. The following table summarizes some relevant studies in previous years [4].

Table 1: Cases of CRISPR gene editing applied in iPSC

Gene-editing type



Disease model

Cell therapy

CRISPR-KO disease modeling

Immunodeficiency, centromeric instability, and facial abnormalities (ICF) syndrome



Dominant dystrophic epidermolysis bullosa(DDEB)



Tangier disease (TD)



Atrial fibrillation (AF)



CRISPR-KO regain normal function

Fragile X syndrome (FXS)



Duchenne muscular dystrophy (DMD)


CRISPR-KI mutation repair

Myositis ossificans progressiva (MOP)



Chronic granulomatous disease (CGD)


Amyotrophic lateral sclerosis (ALS)

SOD1 and FUS


Frontotemporal dementia






Hypertrophic cardiomyopathy (HCM)



CRISPR combined with piggyBAC



Brugada syndrome



Huntington's disease(HD)



Hereditary motor sensory neuropathy (HMSN)



Tetrahydrobiopterin (BH4) deficiency





Polycythemia vera (PV), α 1-antitrypsin (AA T) deficiency



CRISPRi gene knockdown

Long QT syndrome(LQTS)



Are you moved with these cases and interested in CRISPR-mediated iPSC construction? I have to say, even though there are so many cases, the construction of gene-editing iPSC is not always simple: iPSC culture is complex, and a little mistake may cause cell differentiation and loss of stemness, so the construction process needs to be much more careful and time-consuming. In addition, the transfection and monoclonal isolation of iPSC are also difficult, requiring lots of experience and patience. Ubigene has rich experience in gene-editing iPSCs. For iPSCs from different sources, at least three transfection methods are tested to ensure transfection efficiency. In addition, the unique EZ-editor™ single-cell clone validation technology enables rapid high-throughput positive clone screening, making iPSC gene-editing easier!


[1] Romano E, Trionfini P, Ciampi O, et al. Generation of PKD1 mono-allelic and bi-allelic knockout iPS cell lines using CRISPR-Cas9 system[J]. Stem Cell Research, 2020, 47: 101881.

[2] Ou Z, Niu X, He W, et al. The combination of CRISPR/Cas9 and iPSC technologies in the gene therapy of human β-thalassemia in mice[J]. Scientific reports, 2016, 6(1): 1-13.

[3] Kang H J, Minder P, Park M A, et al. CCR5 disruption in induced pluripotent stem cells using CRISPR/Cas9 provides selective resistance of immune cells to CCR5-tropic HIV-1 virus[J]. Molecular Therapy-Nucleic Acids, 2015, 4: e268.

[4] Ben Jehuda R, Shemer Y, Binah O. Genome editing in induced pluripotent stem cells using CRISPR/Cas9[J]. Stem Cell Reviews and Reports, 2018, 14(3): 323-336.

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