Generation of knockout iPSCs using CRISPR-Cas9 genome editing
Pietro De Camilli, Nisha Mohd Rafiq
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
Steps
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.
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 0h 30m 0s at 37°C.
Run the sample on the gel, excise the band, perform a gel extraction, and measure the concentration on the Nanodrop.
Anneal and phosphorylate gRNA.
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°C0h 30m 0s95°C``0h 1m 0s85°C0h 1m 0s75°C0h 1m 0s65°C0h 1m 0s55°C0h 1m 0s45°C``0h 1m 0s35°C0h 1m 0s25°C0h 1m 0s
Ligate the gRNA and PX458.
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 0h 5m 0s at Room temperature, then put On ice to chill.
Transformation
Add 1µL of the ligation to Oneshot Stable3 bacteria.
Incubate On ice for 0h 30m 0s.
Heat shock in the 42°C water bath for 0h 0m 45s.
Place On ice for 0h 2m 0s.
Add 100µL of super optimal broth with catabolite repression (SOC), and place on 37°C shaker for 1h 0m 0s.
Spread all the bacteria on an Ampicillin plate.
Transfection
Dissociate 60-70% confluent iPSCs in a 6-well plate according to Method section.
Transfect cells with 3µg 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 10micromolar (µM) Y-27632 (rock inhibitor).
Cell Sorting
Select GFP-positive cells by Fluorescence-activated cell sorting (FACS) 48h 0m 0spost-transfection
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
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.
