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A fast method to reprogram and CRISPR/Cas9 gene editing from erythroblasts.
Uirá Souto Melo1, Felipe de Souza Leite1, Silvia Costa1
1Department of Genetics and Evolutionary Biology, Human Genome and Stem Cell Research Center, Biosciences Institute, University of São Paulo (USP), São Paulo, SP 05508-900, Brazil.
Stem Cell Research
|July 18, 2018
Summary
Scientists efficiently reprogrammed blood cells into human induced pluripotent stem cells (hiPSC) and simultaneously edited genes using CRISPR/Cas9. This faster, less invasive method creates gene-edited hiPSC from blood in about 5 weeks.
Area of Science:
- Stem Cell Biology
- Gene Editing Technologies
- Hematopoietic Stem Cells
Background:
- Simultaneous reprogramming and gene editing into human induced pluripotent stem cells (hiPSC) from fibroblasts is a recent advancement.
- Current methods are often multi-step and time-consuming.
Purpose of the Study:
- To establish an efficient one-step procedure for simultaneous reprogramming and CRISPR/Cas9 gene editing from erythroblasts.
- To compare the efficiency and time-frame with fibroblast-based methods.
- To assess the safety and accuracy of the gene editing process.
Main Methods:
- Utilized erythroblasts as the starting material for reprogramming.
- Performed CRISPR/Cas9 gene editing concurrently with the reprogramming process.
- Cultured and analyzed resulting human induced pluripotent stem cell colonies for mutations and off-targets.
Main Results:
- Successfully generated human induced pluripotent stem cell colonies with targeted CAPN1 mutations (in-frame and out-of-frame) in one or both alleles.
- Confirmed no off-target mutations in the edited cell lines.
- Completed the entire process from blood collection to mutated hiPSC generation in approximately 5 weeks.
Conclusions:
- Simultaneous reprogramming and gene editing from erythroblasts is an efficient and rapid alternative to fibroblast-based methods.
- This approach offers a less invasive procedure (blood draw vs. skin biopsy) and significantly reduces the overall time for generating gene-edited hiPSC.
- The method is precise, with no identified off-target mutations, making it a promising tool for regenerative medicine and disease modeling.
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