Demonstrated Protocol: CRISPR-Cas9 Genome Editing In Human Induced Pluripotent Stem Cells

Optimizing results in Essential 8 or KnockOut Serum Replacement media systems
 

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Genome editing in human induced pluripotent stem cells (iPSCs) grown in Essential 8 Medium

Cell culture

Gibco™ Human Episomal iPSC Line is cultured in Gibco Essential 8™ Medium on tissue culture dishes coated with Gibco Geltrex™ LDEV-Free, hESC-Qualifi ed, Reduced Growth Factor Basement Membrane Matrix. After thawing, cells are passaged 2–3 times using the recommended protocols. One day prior to transfection, cells are plated as follows:

1. Wash cells grown in a 60 mm plate with 3 mL Gibco DPBS.
2. Add 1 mL Gibco TrypLE™ Express Enzyme and swirl to cover the entire surface. Incubate for 2–3 minutes in a 37°C humidified incubator. Note: Cells should still attach to plate at this point.
3. Aspirate TrypLE Express Enzyme gently. Add 3 mL Essential 8 Medium and dislodge cells into a single-cell suspension by pipetting up and down a few times.
4. Count cells using the Invitrogen Countess™ II Automated Cell Counter or hemocytometer.
5. For transfection with Invitrogen Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (refer to Knockout by transfection of GeneArt Platinum Cas9 Nuclease and guide RNA (gRNA)), plate 4 x 10⁴ cells in a 24-well format in 500 μL of Essential 8 Medium containing 10 μM ROCK inhibitor and Gibco RevitaCell™ Supplement, then allow cells to recover overnight.
6. For the Invitrogen Neon™ Transfection System, refer to Knockout by electroporation of RNP using the Neon Transfection System and Knockout by electroporation of gRNA and GeneArt CRISPR Nuclease mRNA.

Designing and generating gRNA by in vitro transcription

Use the Invitrogen GeneArt™ CRISPR Search and Design Tool, to search our database of >600,000 predesigned gRNA sequences specific to every gene in the human and mouse genomes. GeneArt predesigned gRNAs are optimized for gene knockout and typically target the first 3 transcribed exons per gene.

Generate your DNA template containing the T7 promoter and the gRNA sequence using the Invitrogen GeneArt Precision gRNA Synthesis Kit.

Knockout by transfection of GeneArt Platinum Cas9 Nuclease and guide RNA (gRNA)

Transfection of gene-specific gRNA and Invitrogen GeneArt Platinum™ Cas9 Nuclease complexes can be used for efficient gene knockout in iPSCs [1,2]. Described below is an optimized transfection protocol utilizing Lipofectamine CRISPRMAX Cas9 Transfection Reagent.

1. On the day of transfection, cells should be ~30% confluent (do not change the cell medium at this point).
2. Prepare Cas9–gRNA ribonucleoprotein (RNP) complex and transfection reagent in separate tubes:

Tube 1 mixture will be stable up to 2 hours at room temperature. Always make tube 1 first.

For optimal performance, incubate tube 2 for 5 min (longer incubation will lead to a decrease in transfection efficiency).

3. Add contents of tube 1 to tube 2, briefly vortex, and incubate the resulting complex for 10–15 min at room temperature (the order of addition is important).
4. Add 55 μL complex to one well of a 24-well plate containing cells.
   
5. Six hours posttransfection, replace the medium with 1 mL of fresh iPSC culture medium without ROCK inhibitor for each well of a 24-well plate and place the cell culture dish back into the incubator.
   
6. After 48 hours posttransfection, wash the cells once with 500 μL PBS and analyze cleavage efficiency using the Invitrogen GeneArt™ Genomic Cleavage Detection Kit.

Note: We recommend that users optimize transfection conditions for their specific cell type. We optimize our conditions using our in-house HPRT gRNA control, which is available for purchase as custom gRNA (to order, contact us at GEMServices@thermofisher.com). Using the protocol described here for Lipofectamine CRISPRMAX reagent, we get >50% cleavage efficiency with the HPRT control in the Gibco Human Episomal iPSC Line.

Knockout by electroporation of RNP using the Neon Transfection System

1. On the day of electroporation, detach cells using TrypLE Express Enzyme solution and then resuspend cells in growth medium prior to cell counting as described in Cell culture
   
2. Prepare electroporation complex by adding 0.5 μL of Cas9 nuclease (3 μg/μL) and 300 ng of gRNA (volume of gRNA should be 0.5 μL or less) to 5 μL of Resuspension Buffer R. Mix gently.
3. Incubate the complex at room temperature for 10 minutes.
4. Meanwhile, transfer 1 x 10⁶ cells to a sterile test tube and centrifuge at 300 x g for 5 minutes.
5. Aspirate the supernatant and resuspend the pellet in 1 mL of PBS without Ca²⁺ and Mg²⁺, then centrifuge at 300 x g for 5 minutes.
6. Carefully and completely aspirate the PBS, being careful to not disturb the cells.
7. Resuspend the cells in 50 μL Resuspension Buffer R.
8. Transfer 5 μL of resuspended cells to the 6 μL of Cas9–gRNA complexes prepared in steps 2 and 3 Mix gently.
9. Pipette 10 μL of the cell suspension into the Neon™ tip and electroporate with protocol 7 (1,200 V, 30 ms, 1 pulse). Be careful to not introduce bubbles.

Note: We recommend that users optimize electroporation conditions for the Neon Transfection System for their specific cell type. We optimize our conditions using our in-house HPRT gRNA control, which is available for purchase as custom gRNA (to order, contact us at GEMServices@thermofisher.com). Using the protocol described here for the Neon Transfection System, we get >80% cleavage efficiency with the HPRT control in the Gibco Human Episomal iPSC Line.

10. Immediately transfer the electroporated cells into a 24-well plate containing 0.5 mL of growth medium containing 10 μM ROCK inhibitor or 1X RevitaCell Supplement. Incubate the cells in a humidified 37°C, 5% CO₂ incubator and change medium the next day. Analyze the cells 48–72 hours after electroporation.
11. Harvest cells and measure cleavage efficiency using the GeneArt Genomic Cleavage Detection Kit, Sanger sequencing, or next-generation sequencing.

Knockout by electroporation of gRNA and GeneArt CRISPR Nuclease mRNA

The cell harvesting and electroporation method can be followed as described above (Cell culture and Knockout by electroporation of RNP using the Neon Transfection System, respectively), except for the dosage of RNA and program of the Neon system. When using gRNA and Invitrogen GeneArt CRISPR Nuclease mRNA, 200 ng of in vitro–transcribed gRNA and 1 μg of GeneArt CRISPR Nuclease mRNA are used per well in a 24-well format. In our hands, we found that out of the 24 preprogrammed optimization conditions of the Neon Transfection System, protocol 17 (850 V, 30 ms, 2 pulses) gave the best cleavage efficiency as detected using the GeneArt Genomic Cleavage Detection Kit.

Genome editing in human iPSCs grown in KnockOut Serum Replacement media systems

Cell culture

Feeder-dependent Gibco Human Episomal iPSC Line is cultured on mitotically inactivated MEF feeder cells (EMD Millipore, Cat. No. PMEF-H) in human ESC (hESC) medium containing 20% Gibco KnockOut™ Serum Replacement, 10 μM MEM Non-Essential Amino Acids Solution, 55 μM 2-Mercaptoethanol, and 4 ng/ mL FGF-Basic in DMEM/F-12. iPSC cultures are maintained in a 5% CO₂, 37°C humidified incubator with daily media changes and passaged regularly using Gibco Collagenase Type IV.

Feeder-free adaptation

Feeder-dependent iPSCs are grown to 80% confluency prior to harvesting with collagenase. Following removal of the cell clusters from the feeder layer, cell clusters are gravity-sedimented to prevent MEF contamination. The cell clusters are then seeded on tissue culture plates coated with Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix in MEF-conditioned medium supplemented with 4 ng/mL FGF-Basic. MEFconditioned medium is produced using MEF feeder cells in hESC medium that is harvested on 7 continuous days, sterile-filtered, and frozen until usage. The cultures are allowed to reach 80–90% confluence. The day prior to transfection, cultures should be inspected for signs of differentiation and any contamination from differentiated cells removed via microdissection.

Harvesting cells for transfection and electroporation

1. One day prior to transfection, rinse cells once with DPBS and then incubate with TrypLE Express Enzyme for about 3 min.
2. Monitor the digestion process periodically under the microscope. When gaps and cracks begin to appear within the large colonies, immediately remove TrypLE Express Enzyme and add 3 mL of growth medium.
3. To avoid cell death, gently dislodge cells into small clumps consisting of approximately triplets of cells. Monitor this step under a microscope. Count an aliquot of cells using the Countess II Automated Cell Counter or hemocytometer.
4. Centrifuge cells at 800 rpm for 3 minutes, then carefully aspirate medium and wash cell pellet with DPBS.
5. Plate 4–5 x 10⁴ cells in a 24-well tissue culture plate with medium containing 10 μM ROCK inhibitor and 1X RevitaCell Supplement, then allow cells to recover overnight.
6. Use 1 x 10⁵ cells for electroporation with the Neon Transfection System (Knockout by electroporation of RNP using the Neon Transfection System and Knock-in by electroporation of RNP and ssDNA using
   the Neon Transfection System).

Knockout by transfection of gRNA and GeneArt CRISPR Nuclease mRNA

1. Prepare dilutions of RNA and transfection reagent in separate tubes:

Mix well and incubate for 5 min at room temperature.

Mix well and incubate for 5 min at room temperature.

2. Add contents of tube 1 to tube 2, briefly vortex, and incubate the complex for 10–15 min at room temperature (the order of addition is important).
3. Add the entire complex to one well of a 24-well plate containing cells.
4. Six hours posttransfection, replace medium with 1 mL of fresh iPSC culture medium without ROCK inhibitor and return the plate to the incubator.
5. After 48–72 hours posttransfection, wash the cells once with 500 μL PBS and analyze the cleavage efficiency by using the GeneArt Genomic Cleavage Detection Kit.

Knock-in by transfection of gRNA, GeneArt CRISPR Nuclease mRNA, and single-stranded DNA (ssDNA)

Transfections are performed in a similar fashion as described in Knockout by transfection of gRNA and GeneArt CRISPR Nuclease mRNA. For single-nucleotide changes, 10 pmol of 100 bp ssDNA can be added along with gRNA and GeneArt CRISPR Nuclease mRNA (see Knockout by transfection of gRNA and GeneArt CRISPR Nuclease mRNA, step 1).

Knockout by electroporation of RNP using the Neon Transfection System

Electroporation protocol is similar to the procedure described in Knockout by electroporation of RNP using the Neon
Transfection System.

Knock-in by electroporation of RNP and ssDNA using the Neon Transfection System

Electroporation is performed in a similar fashion as described in Knockout by electroporation of RNP using the Neon
Transfection System. For gene knock-ins, 10 pmol of oligo (97-base ssDNA) is added along with gRNA and Cas9 protein. The total volume of Cas9 protein, gRNA, and ssDNA should not increase more than 1 μL per electroporation reaction. The best electroporation condition we observed for knock-ins on the Neon Transfection System was optimization protocol 21 (1,300 V, 10 ms, 3 pulses).

1. Liang X, Potter J, Kumar S et al. (2015) Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol 208:44–53.
2. Yu X, Liang X, Xie H et al. (2016) Improved delivery of Cas9 protein/gRNA complexes using Lipofectamine CRISPRMAX. Biotechnol Lett 38:919–929.