Page 95 - PCC08

Germline genome editing
DNA editing of germline can be performed in
1. Stem cells
• Pluripotent stem cells to be differentiated into gametes
to further give rise to an embryo (in vitro gametogenesis
–IVG)
• Spermatogonial stem cells
2. Zygotes or Embryos
Workflow
PCC Genetics - ESHRE BCN 2018
Human Pluripotent Stem Cell Gene-Editing
(1) In silico design of the TALE array or CRISPR guide RNA.
(2) Cloning and construction of nuclease and guide RNA vectors, either as DNA, mRNA, or
protein
(3) Transfection or transduction of single-cell hPSCs.
(4) Cell selection (FACS or antibiotics)
(5) hPSC colonies are picked and expanded
(6) Genomic DNA analysis and targeted clone recovery and expansion
Genome Editing in hPSC: Approaches, Pitfalls and solutions PCC Genetics - ESHRE BCN 2018
Hendriks, 2016
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