Feb 22, 2025
CRISPR/Cas9 gene editing in hiPSC
- 1UCL / The Francis Crick Institute
Protocol Citation: Laura Mahoney 2025. CRISPR/Cas9 gene editing in hiPSC. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk9xxdv5r/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: February 21, 2025
Last Modified: February 22, 2025
Protocol Integer ID: 123201
Abstract
This protocol is adapted from Skarnes et al., 2019 and describes the steps to follow for knocking-in point mutation via CRISP/Cas9 in humain inducible pluripotent cells (hiPSC)
A. Pre-nucleofection
A. Pre-nucleofection
Resuspend the sgRNA overnight at 4C in TE buffer at a concentration of4 µg/µL
Resuspend the Alt-R HDR modified ssODN overnight in D-PBS at a concentration of 200 pmol/ul
For each nucleofection, prepare 6ml StemFlex + Revitacell or Rock inhibitor and coat 1x 6-well plate with geltrex/synthemax/matrigel for 01:00:00 at 37 °C
For each nucleofection, prepare complete P3 solution by adding 20 µL of Supplement to 90 µL of P3 solition in a 1.5ml Eppendorf
B. Pre-assembly of Cas9 RNP
B. Pre-assembly of Cas9 RNP
For each nucleofection, mix 4 µL of sgRNA (16 µg total) and 2 µL of recombinant Cas9 protein (20 µg total), in 1.5ml Eppendorf tube
Incubate at RT for 00:30:00 to 00:45:00 . Note: begin harvesting iPSC
after 15mins
Immediately prior to nucleofection, add 1 µL (200 pmol ) of Alt-R HDR modified ssODN to the pre-assembled Cas9 RNP.
C. Delivery of Cas9 RNP and ssODN using Nucleofector
C. Delivery of Cas9 RNP and ssODN using Nucleofector
Switch on the nucleofector unit and select “X-unit” and single cuvette. Use the “Primary Cell P3” program and set the pulse code to “CA-137”
Collect the iPSC from an 80% confluent well with pre-warmed accutase. Count cells and pellet 8 x 105 cells by centrifuging at 300 x g, 20°C, 00:05:00 and aspirate supernatant.
While the cells are spinning, add the 1 µL of ssODN to the pre-assembled Cas9 RNP Eppendorf.
After pelleting cells and supernatant removed, gently resuspend the cell pellet in 100 µL complete P3 Solution and transfer the cells to the Eppendorf containing Cas9 RNP+ssODN. Transfer the cell suspension to the nucleocuvette. Tap the cuvette on a flat surface to eliminate bubbles and nucleofect immediately using the “Primary Cell P3” program and “CA-137” pulse code.
Immediately after the nuclefection, transfer the cells into one well of pre-coated, warm
6-well plate containing 2-3 ml of Stemflex + revitacell/Rock. Inh.
Add HDR enhancer to a final concentration of 30 micromolar (µM) (1:100 from the 3mM stock
solution). Culture the cells in a 32 °C incubator (cold shock for 2 days: important for homozygous gene editing).
Change media the next day to StemFlex without revitacell/rock inh and HDR enhancer
Return cells to 37 °C incubator on day 3
D. Plating single cells to isolated clones
D. Plating single cells to isolated clones
Once the cells in the 6-well plate are 80% confluent, dissociate them with accutase into a single cell suspension, centrifuge at 210 x g, 20°C, 00:05:00 . Remove supernatant and resuspend cell pellet in StemFlex + revitacel/rock.inh
Count the cells and plate between 800 – 1000 single cells in geltrex/synthemax/matrigel-coated 10cm
petri dishes containing 10ml Stemflex + revitacel/rock inh.
Transfer cells to 37 °C incubator.
Change the media the next day and daily with Stemflex to remove revitacell/rock inhibitor
E. Picking single cell-derived clones
E. Picking single cell-derived clones
With the aid of a dissecting microscope and a p200, individual colonies are picked and transferred into 2x 96-well plates, one for colonies expansion, the other for DNA lysis and genotyping.
For each 96 colonies to be picked, prepare 1x geltrex coated 96-well and 15ml of Stemflex+Rock inh/revitacell and warm to RT in the hood.
Use the p200 tip to break apart the colony from the plate and collect the cell fragments in a volume of 50 µL of media. Transfer each colony to a well of a U-bottom 96-well plate.
Once 96 colonies are picked, add 150 µL of Stemflex+rock inh/revitacell to each well of the U-bottom plate with a multichannel.
With the use of the multichannel set to 100ul, pipette cells up and down 10 times in the U-bottom plate. Transfer 100 µL of the cell suspension to geltrex coated 96-well plate (for colony expansion) and 100 µL to 96-well plate for lysis and DNA extraction.
The following day, replace the media in the expansion 96-well plate with fresh Stemflex without revitacell/rock inh.
F. Genomic DNA extraction from 96-well plates
F. Genomic DNA extraction from 96-well plates
For each 96-well plate to be lysed, add 10 mg of proteinase K powder (Sigma-Aldrich, P6556) to 10 mL of lysis buffer (50 millimolar (mM) KCl, 10 millimolar (mM) Tris-HCl pH 8, 2 millimolar (mM) MgCl2, 0.45 % volume (v/v) NP40, 0.45 % volume (v/v) Tween20).
With a multichannel pipettor, remove media from confluent wells of the Synthemax-coated 96-well plate and wash each well once with 150 µL room temperature D-PBS.
Add 100 µL of lysis buffer containing 1 mg/ml proteinase K, seal the 96-well plate andcincubate at 60 °C for at least 04:00:00 .
While the plate is still warm, transfer lysates to a 96-well PCR plate, seal the plate and heat inactivate the proteinase K by incubating the plate at 95 °C for 00:10:00 in a thermocycler.
While the samples are warm, prepare a 1:20 dilution of the lysates in water for setting
up PCR reactions. Store the undiluted lysates at -80 °C . The diluted samples
can be kept at 4 °C for a few days.