TITLE: Generation of knockout iPSCs using CRISPR-Cas9 genome editing
AUTHORS: Nisha Mohd Rafiq, Pietro De Camilli

[DESCRIPTION]
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).

[STEPS]
SECTION: Molecular cloning
1. 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.
SECTION: Molecular cloning
2. 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).
SECTION: Molecular cloning
3. Cloning reagents are purchased from Thermo Scientific Fisher, unless stated otherwise.
SECTION: Digest PX458.
4. Prepare the following reaction mixture:

 

SECTION: Digest PX458.
5. Incubate the sample for 30 min at 37 °C.

SECTION: Digest PX458.
6. Run the sample on the gel, excise the band, perform a gel extraction, and measure the concentration on the Nanodrop.
SECTION: Anneal and phosphorylate gRNA.
7. Prepare the following reaction mixture:
 
 
SECTION: Anneal and phosphorylate gRNA.
8. Set up the following parameters on the PCR machine:

 37 °C  30 min
 95 °C1 min
 85 °C  1 min
75 °C  1 min 
65 °C 1 min
55 °C  1 min 
45 °C1 min
35 °C  1 min
25 °C  1 min    

SECTION: Ligate the gRNA and PX458.
9. Prepare the following reaction mixture: 
 
 
SECTION: Ligate the gRNA and PX458.
10. Incubate the transformation for 5 min at Room temperature, then put on ice to chill.

SECTION: Transformation
11. Add 1 µL of the ligation to Oneshot Stable3 bacteria.

SECTION: Transformation
12. Incubate on ice for 30 min.

SECTION: Transformation
13. Heat shock in the 42 °C water bath for 45 s.

SECTION: Transformation
14. Place on ice for 2 min.

SECTION: Transformation
15. Add 100 µL of super optimal broth with catabolite repression (SOC), and place on 37 °C shaker for 60 min.

SECTION: Transformation
16. Spread all the bacteria on an Ampicillin plate.
SECTION: Transfection
17. Dissociate 60-70% confluent iPSCs in a 6-well plate according to Method section.
SECTION: Transfection
18. Transfect cells with 3 µg PX458-gRNA using P3 Primary Cell 4D-Nucleofector™ (Lonza) using CA-137 parameter according to manufacturer’s protocol. 

SECTION: Transfection
19. Plate nucleofected cells in geltrex-coated dishes in E8 Flex media containing 10 micromolar (µM) Y-27632 (rock inhibitor).
SECTION: Cell Sorting
20. Select GFP-positive cells by Fluorescence-activated cell sorting (FACS) 2880 minpost-transfection

SECTION: Cell Sorting
21. After sorting, plate the cells in Geltrex-coated dish in E8 Flex media containing Y-27632 (rock inhibitor). 
SECTION: Cell Sorting
22. Replace the media with fresh E8 Flex media the next day.
SECTION: Immunoblotting and Sanger sequencing
23. Check wells each day to identify wells with a single colony. Split and expand each clonal well when possible.
SECTION: Immunoblotting and Sanger sequencing
24. Screen colonies for target protein using immunoblotting or sanger sequencing.
SECTION: Immunoblotting and Sanger sequencing
25. 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).
SECTION: Immunoblotting and Sanger sequencing
26. Expand and freeze KO lines.