Using CRISPR Cas9 genetic editing tool to repair mutation in induced pluripotent stem cells from a patient with retinis pigmantosa

1. Hashemite University
Genetic Repair of Retinitis Pigmentosa in Patient Derived Stem Cells
- Presented by : Belal Asa’d

2. Precision Medicine: Genetic Repair of Retinitis
Pigmentosa in Patient Derived Stem Cells

3. Introduction
• Retinitis pigmentosa (RP)
• Inherited ( X-linked ), degenerative eye disease
• causes severe vision impairment
• progressive degeneration of the rod
photoreceptor cells in the retina

4. • The disease caused of point mutation
• C.3070 (T) at ORF15 of Retinis Rigmentosa
GTPase Regulator gene
• Stop codon stop protein synthesis
• TAG - - - - - < GAG
Stop codon- - - < Glutamate

5. Materials and Methods
• 2 technologies used in this research paper
• Genetic editing tool :
CRISPR Cas-9 ** system and
• Stem cells
Induced pluripotent stem cells
** clustered regulatory-interspaced short palindromic repeats

6. Making stem cells
• Skin-punch biopsy taken (own patient )
• Fibroblast cells taken.. Generation ability to
connective tissue
• Transcription factors added
• Induced pluripotent stem cells form (iPSCs)
• But.. iPSCs still have point mutation (TAG)

7. Skin-punch biopsy

8. Stem cells typed
• Pluripotent Stem Cells
- Ability to generate any type of cells
- Ex: Embryonic stem cells or iPSCs
- ( Embryo ) ( Adult )
• Multipotent Stem Cells
- Generate certain type of cells
- Ex: Hematopoietic stem cells

9. Why we don’t take stem cells from
embryos directly
• Ethical issues
• Risk of immuno-mediated rejection

10. Testing Pluripotency
• Test Pluripotency markers (Sox2)
• Testing Ability to for forming three layers

11. Crispr Cas-9 editing tool

12. CRISPR Cas9
• Composed of 2 RNA guides + Cas9 endonuclease
• Transfection of iPSCs with expression vector Cas9
• Efficiency of correct cuts was 23%
• Homology-directed gene repair (HDR) completed
sequence after double strand break (DSB)
• Correction percent of the mutation (G>T) of 223
cells transfected with Cas9 was 13%

13. Crispr Cas9 technology
• https://www.youtube.com/watch?v=ow0X8W
ifP08

14. Results

15. Autofluorescence imaging (A+B) + Optical coherence
tomography (C+D)

16. Skin-punch biopsy culture and fibroblast(E) + Alkaline phosphatase
stain (F) + Immunohistochemistry and fluorescence microscopy (G,H)

17. Injection of iPSCs into a sever combined
immunodeficiency mouse forming 3 germ line layers

18. Crispr Cas9 and choosing gRNA

19. Insertion of gRNA into expression
vector alongside Cas9 nuclease

20. PCR products analysis by 2 different polymerases show gRNA 58
is more precise in CRISPR Cas9 cleavage of target site mutation

21. Sanger sequencing – Dideoxynucleotide of GTPase Regulator gene in
Retinis Pigmentosa patient sample (Top) and Transfected cells with
gRNA-Cas9 expression vector (Bottom)

22. Deep sequencing for control and Cas9 technology

24. Conclusion
• The efficiency of cleavage of target site 3070 G>T for
CRISPR Cas9 – (g58 - gRNA) was 23% out of 293 cell
line
• Correction Rate of 23% those cells due to cell
replication proofreading machinery was 13%
according homology-directed gene repair (HDR)
• The next step is to convert corrected iPSCs to retinal
cells and transplant it in retina of the same patient

25. Reference
• Bassuk, Alexander G., Andrew Zheng, Yao Li, Stephen H.
Tsang, and Vinit B. Mahajan. "Precision Medicine: Genetic
Repair of Retinitis Pigmentosa in Patient-Derived Stem
Cells." Sci. Rep. Scientific Reports 6 (2016)