| Chapter title |
CRISPR/Cas-Mediated Knock-in of Genetically Encoded Fluorescent Biosensors into the AAVS1 Locus of Human-Induced Pluripotent Stem Cells.
|
|---|---|
| Chapter number | 422 |
| Book title |
Induced Pluripotent Stem Cells and Human Disease
|
| Published in |
Methods in molecular biology, September 2021
|
| DOI | 10.1007/7651_2021_422 |
| Pubmed ID | |
| Book ISBNs |
978-1-07-162584-2, 978-1-07-162585-9
|
| Authors |
Stellon, David, Tran, Minh Thuan Nguyen, Talbot, Jana, Chear, Sueanne, Khalid, Mohd Khairul Nizam Mohd, Pébay, Alice, Vickers, James C, King, Anna E, Hewitt, Alex W, Cook, Anthony L, Vickers, James C., King, Anna E., Hewitt, Alex W., Cook, Anthony L. |
| Abstract |
Genetically encoded fluorescent biosensors (GEFBs) enable researchers to visualize and quantify cellular processes in live cells. Induced pluripotent stem cells (iPSCs) can be genetically engineered to express GEFBs via integration into the Adeno-Associated Virus Integration Site 1 (AAVS1) safe harbor locus. This can be achieved using CRISPR/Cas ribonucleoprotein targeting to cause a double-strand break at the AAVS1 locus, which subsequently undergoes homology-directed repair (HDR) in the presence of a donor plasmid containing the GEFB sequence. We describe an optimized protocol for CRISPR/Cas-mediated knock-in of GEFBs into the AAVS1 locus of human iPSCs that allows puromycin selection and which exhibits negligible off-target editing. The resulting iPSC lines can be differentiated into cells of different lineages while retaining expression of the GEFB, enabling live-cell interrogation of cell pathway activities across a diversity of disease models. |
X Demographics
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 2 | 100% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Members of the public | 2 | 100% |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 17 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Researcher | 4 | 24% |
| Other | 1 | 6% |
| Student > Bachelor | 1 | 6% |
| Lecturer | 1 | 6% |
| Student > Master | 1 | 6% |
| Other | 1 | 6% |
| Unknown | 8 | 47% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 3 | 18% |
| Pharmacology, Toxicology and Pharmaceutical Science | 1 | 6% |
| Agricultural and Biological Sciences | 1 | 6% |
| Immunology and Microbiology | 1 | 6% |
| Economics, Econometrics and Finance | 1 | 6% |
| Other | 1 | 6% |
| Unknown | 9 | 53% |