Mar 19, 2024
Generation of knockout iPSCs using CRISPR-Cas9 genome editing
- Nisha Mohd Rafiq1,
- Pietro De Camilli2,3,4,5
- 1University of Tuebingen;
- 2Department of Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA.;
- 3Department of Cell biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.;
- 4Program in Cellular Neuroscience, Neurodegeneration and Repair.;
- 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA.
Protocol Citation: Nisha Mohd Rafiq, Pietro De Camilli 2024. Generation of knockout iPSCs using CRISPR-Cas9 genome editing. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqnr33gk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 12, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 96918
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000580
Abstract
This protocol describes the genetic modification of induced pluripotent cells (iPSCs) using CRISPR-Cas9, including synthesis of gRNA plasmids, transfection, selection of clones, and sequencing of genomic DNA to confirm knockout generation. The steps described in this protocol is based on Skarnes et al. (2019) and Fernandopulle et al. (2018).
Attachments
Materials
Nucleic acid reagents:
- pSpCas9(BB)-2A-GFP (PX458)addgeneCatalog #48138
- Guide RNAs (gRNAs) (Designed in CHOPCHOP and ordered from Integrated DNA Technologies)
1. Prepare the following reaction mixture:
| A | B | |
| Fast AP | 3 µl | |
| Fast Digest BBS1 | 3 µl | |
| 1x Fast Digest Green | 6 µl | |
| PX458 | 5 µg | |
| H2O | (up to 60 µl) |
2. Prepare the following reaction mixture:
| A | B | |
| Sense gRNA (100 µM stock) | 1 µl | |
| Antisense gRNA (100 µM stock) | 1 µl | |
| T4 PNK Buffer | 1 µl | |
| ATP (10 mM) | 1 µl | |
| T4 PNK | 0.5 µl | |
| H2O | 5.5µl |
3. Prepare the following reaction mixture:
| A | B | |
| PX458 Vector | 50 ng | |
| gRNA (1:200 Stock) | 1 µl | |
| 2X Quick Buffer* | 5 µl | |
| sH2O | 1.5 µl | |
| Quick Ligase* | 1 µl | |
| 11 µl |
*Quick Ligation™ Kit (New England Biolabs).
Molecular cloning
Molecular cloning
Design gRNAs using CHOPCHOP (https://chopchop.cbu.uib.no/). Specific gRNAs used in this study can be found in the method section of our manuscript.
Order gRNAs as complementary single stranded oligonucleotides (Integrated DNA Technologies). The indicated overhanging nucleotides should be included to allow for cloning (Method section of our manuscript).
Cloning reagents are purchased from Thermo Scientific Fisher, unless stated otherwise.
Digest PX458.
Digest PX458.
30m
Prepare the following reaction mixture:
| A | B | |
| Fast AP | 3 µl | |
| Fast Digest BBS1 | 3 µl | |
| 1x Fast Digest Green | 6 µl | |
| PX458 | 5 µg | |
| H2O | (up to 60 µl) |
Incubate the sample for 00:30:00 at 37 °C .
30m
Run the sample on the gel, excise the band, perform a gel extraction, and measure the concentration on the Nanodrop.
Anneal and phosphorylate gRNA.
Anneal and phosphorylate gRNA.
38m
Prepare the following reaction mixture:
| A | B | |
| Sense gRNA (100 µM stock) | 1 µl | |
| Antisense gRNA (100 µM stock) | 1 µl | |
| T4 PNK Buffer | 1 µl | |
| ATP (10 mM) | 1 µl | |
| T4 PNK | 0.5 µl | |
| H2O | 5.5µl |
Set up the following parameters on the PCR machine:
- 37 °C 00:30:00
- 95 °C 00:01:00
- 85 °C 00:01:00
- 75 °C 00:01:00
- 65 °C 00:01:00
- 55 °C 00:01:00
- 45 °C 00:01:00
- 35 °C 00:01:00
- 25 °C 00:01:00
38m
Ligate the gRNA and PX458.
Ligate the gRNA and PX458.
5m
Prepare the following reaction mixture:
| A | B | |
| PX458 Vector | 50 ng | |
| gRNA (1:200 Stock) | 1 µl | |
| 2X Quick Buffer* | 5 µl | |
| sH2O | 1.5 µl | |
| Quick Ligase* | 1 µl | |
| 11 µl |
*Quick Ligation™ Kit (New England Biolabs).
Incubate the transformation for 00:05:00 at Room temperature , then put On ice to chill.
5m
Transformation
Transformation
1h 32m 45s
Add 1 µL of the ligation to Oneshot Stable3 bacteria.
Incubate On ice for 00:30:00 .
30m
Heat shock in the 42 °C water bath for 00:00:45 .
45s
Place On ice for 00:02:00 .
2m
Add 100 µL of super optimal broth with catabolite repression (SOC), and place on 37 °C shaker for 01:00:00 .
1h
Spread all the bacteria on an Ampicillin plate.
Transfection
Transfection
Dissociate 60-70% confluent iPSCs in a 6-well plate according to Method section.
Transfect cells with 3 µg PX458-gRNA using P3 Primary Cell 4D-Nucleofector™ (Lonza) using CA-137 parameter according to manufacturer’s protocol.
Plate nucleofected cells in geltrex-coated dishes in E8 Flex media containing 10 micromolar (µM) Y-27632 (rock inhibitor).
Cell Sorting
Cell Sorting
2d
Select GFP-positive cells by Fluorescence-activated cell sorting (FACS) 48:00:00 post-transfection
2d
After sorting, plate the cells in Geltrex-coated dish in E8 Flex media containing Y-27632 (rock inhibitor).
Replace the media with fresh E8 Flex media the next day.
Immunoblotting and Sanger sequencing
Immunoblotting and Sanger sequencing
Check wells each day to identify wells with a single colony. Split and expand each clonal well when possible.
Screen colonies for target protein using immunoblotting or sanger sequencing.
For sanger sequencing, ICE CRISPR analysis tool (https://www.synthego.com/products/bioinformatics/crispr-analysis) was used to analyze the relative contribution of insertions and deletions (INDELS).
Expand and freeze KO lines.
Protocol references
1. Fernandopulle, M.S., R. Prestil, C. Grunseich, C. Wang, L. Gan, and M.E. Ward. 2018. Transcription Factor-Mediated Differentiation of Human iPSCs into Neurons. Curr Protoc Cell Biol. 79:e51.
2. Skarnes, W.C., E. Pellegrino, and J.A. McDonough. 2019. Improving homology-directed repair efficiency in human stem cells. Methods. 164-165:18-28.