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Genome Editing of Human Pluripotent Stem Cells

  • Document # 27084
  • Version 2.0.0
  • Feb 2019

Introduction

The ability of human embryonic stem (ES) and induced pluripotent stem (iPS) cells to self-renew indefinitely and differentiate into all somatic cell types makes them an attractive source of human tissue for regenerative medicine. This potential, combined with recent advances in more efficient and accessible genome editing techniques, has opened the door to a wide range of research areas. Disease-causing mutations can now be introduced or corrected in cell lines to create or rescue disease models; work in this area may also pave the way to correcting disease-causing mutations in vivo.

Figure 1. Experimental Workflow for Human Pluripotent Stem Cell (hPSC) Genome Editing

The guide RNA (gRNA) sequence is designed once a target locus for editing is identified. The ArciTect™ CRISPR-Cas9 ribonucleoprotein (RNP) complex is then prepared and delivered into hPSCs in single-cell suspension using electroporation with or without addition of a donor DNA template (e.g. single-stranded oligodeoxynucleotide [ssODN]). Cells are then plated in hPSC maintenance medium (mTeSR™1 or mTeSR™ Plus) supplemented with CloneR™, to enhance survival of hPSCs plated as single cells. Editing efficiency can be analyzed after 48 - 72 hours using ArciTect™ T7 Endonuclease I Kit (Catalog #76021), via sequencing-based approaches, or by flow cytometry, if the experimental design permits. If generating clonal cell lines is desired, this can be accomplished by limited dilution cloning using mTeSR™1 or mTeSR™ Plus supplemented with CloneR™. Editing efficiency measurements can be used to inform the approximate number of clones for further characterization using sequencing-based approaches.

Design of a CRISPR-Cas9 genome editing experiment is dependent on the experimental goal. If the goal is to understand the general role of a gene in disease, a knockout model is a frequently used approach. Alternatively, one can introduce precise DNA changes that incorporate a single nucleotide variant, genetic “tags”, or transgenes into the genome. These two approaches both require a CRISPR-associated endonuclease protein (i.e. Streptococcus pyogenes Cas9, or SpCas9) and a custom-designed guide RNA (gRNA). The gRNA functions to target Cas9 to the genome. Precise (knock-in) editing also requires addition of a donor DNA template to instruct the cell on the specific genetic changes you wish to incorporate. The exogenous DNA is targeted to the site of interest through incorporation of homologous sequences (also known as homology arms) that flank the target site and can be used as a donor template for repair following Cas9-mediated DNA break formation. This type of experimental approach relies on the homology-directed repair (HDR) pathway, which functions through homologous recombination to incorporate the exogenous sequence at the cleavage site. This is a particularly powerful approach, as it enables researchers to “re-write” the genome in a targeted and specific manner.

Design of a CRISPR-Cas9 genome editing experiment is dependent on the experimental goal. If the goal is to understand the general role of a gene in disease, a knockout model is a frequently used approach. Alternatively, one can introduce precise DNA changes that incorporate a single nucleotide variant, genetic “tags”, or transgenes into the genome. These two approaches both require a CRISPR-associated endonuclease protein (i.e. Streptococcus pyogenes Cas9, or SpCas9) and a custom-designed guide RNA (gRNA). The gRNA functions to target Cas9 to the genome. Precise (knock-in) editing also requires addition of a donor DNA template to instruct the cell on the specific genetic changes you wish to incorporate. The exogenous DNA is targeted to the site of interest through incorporation of homologous sequences (also known as homology arms) that flank the target site and can be used as a donor template for repair following Cas9-mediated DNA break formation. This type of experimental approach relies on the homology-directed repair (HDR) pathway, which functions through homologous recombination to incorporate the exogenous sequence at the cleavage site. This is a particularly powerful approach, as it enables researchers to “re-write” the genome in a targeted and specific manner.

For best results, starting hPSC cultures should be of high quality and moderate density, and largely free from differentiated areas. For complete instructions on culturing high-quality ES and iPS cells, including coating plates and use of mTeSR™1 (Catalog #85850) or (Catalog #05825), refer to the Technical Manual: Maintenance of Human Pluripotent Stem Cells in mTeSR™1 (Document #28315) or mTeSR™ Plus (Document #DX23032), available at www.stemcell.com.

This protocol uses CloneR™ (Catalog #05888), an mTeSR™1/mTeSR™ Plus supplement, which greatly enhances the cloning efficiency and single-cell survival of human pluripotent stem cells (hPSCs). For complete instructions on thawing, preparation, and storage of CloneR™, refer to the Product Information Sheet (Document #DX21725), available at www.stemcell.com or contact us to request a copy.

Preparation of ArciTect™ crRNA and ArciTect™ tracrRNA Stock Solutions

Materials Required

  1. Briefly centrifuge the vials before opening.
  2. Add nuclease-free water to give a final concentration of 200 μM, as outlined in Table 1.

  3. Table 1. Resuspension Volumes for 200 μM* ArciTect™ crRNA or ArciTect™ tracrRNA

    *200 μM is equal to 200 pmol/μL

  4. Mix thoroughly. If not used immediately, aliquot and store at -80°C for up to 6 months. After thawing the aliquots, use immediately. Do not re-freeze.

Electroporation of Human ES and iPS Cells Using the Neon® Transfection System

Materials Required

A. Preparation of Tissue Culture Plates

The following protocol is for electroporation of human ES or iPS cells in a 24-well tissue culture plate. If using other cultureware, adjust volumes accordingly.

  1. Coat a 24-well plate with Matrigel® and bring to room temperature (15 - 25°C) for at least 30 minutes prior to use.
  2. Warm (15 - 25°C) sufficient volumes of mTeSR™1 or mTeSR™ Plus, CloneR™, and ACCUTASE™.
  3. Prepare 5 mL of Single-Cell Plating Medium per transfection by adding CloneR™ to mTeSR™1 or mTeSR™ Plus at a 1 in 10 dilution.
    Example: To prepare 10 mL of Single-Cell Plating Medium, add 1 mL of CloneR™ to 9 mL of mTeSR™1 or mTeSR™ Plus.
  4. Remove Matrigel® from the 24-well plate and replace with 1 mL of Single-Cell Plating Medium per well. Place plate in a 37°C and 5% CO2 incubator.

B. Preparation of Guide RNA (gRNA)

Preparation of RNP and transfection should be performed approximately 24 hours after plating ES or iPS cells.

  1. Prepare 80 μM gRNA by combining crRNA, tracrRNA, and Annealing Buffer in a microcentrifuge tube as indicated in Table 2. Mix thoroughly.
  2. Table 2. Preparation of 80 μM gRNA

  3. In a thermocycler or heating block, incubate gRNA mixture at 95°C for 5 minutes followed by 60°C for 1 minute. Cool to room temperature (15 - 25°C) and place on ice. If not used immediately, store at -80°C for up to 6 months.

C. Preparation of a Single-Cell Suspension

  1. Collect cells during their exponential growth phase.
  2. Use a microscope to visually identify regions of differentiation (if any) in the wells to be passaged. Mark these using a felt tip or lens marker on the bottom of the plate. Remove regions of differentiation by scraping with a pipette tip or by aspiration.
  3. Aspirate the remaining medium from the well and add 1 mL of ACCUTASE™ (for 6-well plate). Incubate the plate at 37°C and 5% CO2 for approximately 5 minutes, or until colonies appear to be dissociated.
  4. Add 2 mL mTeSR™1 or mTeSR™ Plus to each well. Using a pipettor fitted with a 1000 μL tip, gently wash the cells from the surface of the plate by spraying the solution directly onto the colonies. Pipette the suspension up and down 2 - 3 times to break up small aggregates into single cells.
  5. Transfer the cell suspension to a 15 mL conical tube.
  6. Centrifuge at 300 x g for 5 minutes.
  7. Aspirate the supernatant, being careful not to disturb the cell pellet.
  8. Resuspend cells in at least 2 mL of Single-Cell Plating Medium and mix by flicking the tube 2 - 3 times.
  9. Count cells using a hemocytometer or automated cell counter.
  10. Add 3 x 105 cells to a new 15 mL conical tube for each electroporation condition. Centrifuge at 300 x g for 5 minutes.
  11. During centrifugation proceed immediately to section D.

D. Preparation of ArciTect™ CRISPR-Cas9 RNP Complex Mix for Electroporation

  1. To prepare the RNP Complex Mix, combine components in a microcentrifuge tube as indicated in Table 3. Adjust component volumes according to the desired number of transfections. Mix thoroughly.
  2. Table 3. Preparation of RNP Complex Mix

    *If using 3 μg/uL ArciTect™ Cas9-eGFP Nuclease add 1.2 μL of Cas9-eGFP and 3.24 μL of Resuspension Buffer R per 5 μL transfection volume. Note that 1 μg/μL Cas9 Nuclease or Nickase cannot be used for 10 μL Neon® electroporation, as the RNP volume should not exceed 1/10th the total reaction volume.

    NOTE: May require optimization with different cell lines. A 1:2 (shown) to 1:4 molar ratio of Cas9 to guide RNA is recommended.

    NOTE: If two or more guide RNAs are to be used in the experiment (e.g. using ArciTect™ Cas9 Nickase), prepare each RNP complex separately.

  3. Incubate the RNP Complex Mix at room temperature (15 - 25°C) for 20 minutes.
  4. (Optional) Dilute 100 μM ssODN to 20 μM with Resuspension Buffer R as indicated in Table 4.
  5. Table 4. Preparation of Diluted ssODN


  6. After the RNP complex is formed, add 2.5 μL of 20 μM Diluted ssODN or Resuspension Buffer R to each RNP Complex Mix prepared in step 2, according to Table 5.
  7. Table 5. Recommended Electroporation Conditions for Human ES or iPS cells Using a Neon® Device


E. Electroporation of Human ES or iPS Cells with RNP Complex

  1. Aspirate supernatant from the cell pellet prepared in section C, then flick the tube 2 - 3 times to dislodge and break up the pellet.
  2. Resuspend 3 x 105 cells in 7.5 μL of Resuspension Buffer R per electroporation condition (prepared in section C step 10) and pipette up down to mix.
  3. Transfer 7.5 μL of the cell suspension to each 7.5 μL Complete RNP Complex (formed in section D step 4) and pipette up and down to mix.
  4. Using a 10 μL Neon® pipette tip, draw up 10 μL of the mixture, check to see if the capillary is free of bubbles, and place into the electroporation chamber containing 3 mL of Electrolytic Buffer E.

    NOTE: If air bubbles are present in the tip when the cells are electroporated, cell viability and transfection efficiency will be significantly reduced.
  5. Electroporate the mixture using the settings in Table 6.

    NOTE: Refer to the manufacturer’s instructions on electroporation. Electroporation conditions may require optimization for different cell lines.

  6. Table 6. Recommended Electroporation Conditions for Human ES or iPS cells Using a Neon® Device


  7. Immediately after electroporation, transfer cells to the warm (37°C) plate prepared in section A.
  8. Mix by gently rocking the plate back and forth 2 - 3 times.
  9. Incubate the plate at 37°C and 5% CO2.
  10. Perform a full medium change every 24 hours with 0.5 mL of room temperature (15 - 25°C) mTeSR™1 or mTeSR™ Plus.
  11. Incubate the cells for 48 - 72 hours (or up to 7 days if confluency is low) after transfection for genome editing to occur.
  12. Harvest cells for assessment of genome editing efficiency. Genomic DNA can be amplified by PCR using primers flanking the target region and ArciTect™ High-Fidelity DNA Polymerase Kit (Catalog #76026), followed by sequencing of PCR products. Alternatively, .ArciTect™T7 Endonuclease I Kit (Catalog #76021) can be used to assess editing efficiency (% INDEL formation) following PCR amplification. For further information, refer to the Technical Bulletin: Evaluation of Genome Editing (Document #27126), available at www.stemcell.com or contact us to request a copy.

Case Study: Knock-In Optimization Through GFP to BFP Conversion

To demonstrate genetic knockout and knock-in with the ArciTect™ CRISPR-Cas9 system, we targeted the enhanced green fluorescent protein (eGFP) locus in eGFP-tagged WLS-1C iPS and H1 ES cell lines. A single base substitution (196T>C) in the wild-type GFP sequence can shift fluorescence absorption and emission towards the blue spectrum, effectively converting GFP to blue fluorescent protein, or BFP.1 The conversion of GFP to BFP can be used for quantitative assessment of knockout (NHEJ) and knock-in (HDR) frequencies through measuring GFP and BFP fluorescence by flow cytometry.2 The loss of GFP expression indicates genetic knockout (NHEJ) and the conversion of GFP to BFP indicates genetic knock-in of the BFP sequence (Figure 2).

Figure 2. eGFP-tagged hPSC Lines Enable Quantitative Assessment of CRISPR-Cas9 Editing Efficiency

A) Schematic of the eGFP DNA and amino acid sequences, with critical GFP amino acid sequence indicated in green and BFP sequence differences indicated underneath in blue. The crRNA (orange arrow) and single-stranded oligodeoxynucleotide (ssODN) knock-in donor template are shown above and below the sequences, respectively. Ellipses before and after ssODN sequence represent homology arms of 120 base pairs (bp) upstream and 48bp downstream flanking the PAM. The PAM sequence is underlined and silent mutations, to prevent gRNA reannealing and repeated cut-mutation cycles3, are shown in red. (B - D) Representative dot plots of flow cytometry analysis from mock electroporated (B), RNP electroporated (C), and RNP+ssODN electroporated (D) 1C-eGFP hPSCs.


The efficiency of HDR-dependent knock-in editing is considerably lower than knockout since NHEJ is the primary repair pathway in mammalian cells. However, editing efficiency can be optimized through strategic design of both the gRNA and ssODN donor template. Editing efficiency is dependent on the cut-to-mutation distance.3 Since Cas9 cleaves the DNA 3 - 4 base pairs upstream of the PAM sequence, the location of the cut site relative to the desired mutation should be carefully considered. For ssODN donor template design, if the desired knock-in is small (1 - 2 base pairs), the Cas9-gRNA RNP complex may still be able to recognize the sequence and engage in repeated cleavage-mutation cycles until a sufficient Cas9-gRNA-blocking mutation is generated e.g. an INDEL, effectively reducing knock-in editing efficiency. To overcome this, a mutation disrupting the PAM site, or 3 - 4 silent/synonymous mutations (Figure 2A), can be incorporated into the donor DNA template design.3



Figure 3. Efficient genetic knockout and knock-in in hPSCs using the ArciTect™ CRISPR-Cas9 System

H1-eGFP and 1C-eGFP hPSC lines were cultured in mTeSR™1, or adapted to mTeSR™ Plus hPSC maintenance medium for at least two passages prior to initial experiments. (A) Knockout (% GFP- cells) and (B) knock-in (% BFP+ cells) were measured by flow cytometry 72 hours after electroporation with ArciTect™ RNP complexes; n = 3. No significant differences were observed within cell lines.



hPSCs were electroporated with RNP complexes containing GFP crRNA and ssODN (Figure 2A). Knockout and knock-in efficiency were measured 72 hours after electroporation, and we observed up to 53.2% knockout efficiency and up to 3.7% knock-in efficiency (Figure 3). Editing efficiency was equivalent when hPSCs were cultured in either mTeSR™1 or mTeSR™ Plus, however, we did observe cell line-dependent differences in editing efficiency.

Transfection of Human ES and iPS Cells Using the TransIT-X2® Dynamic Delivery System

Materials Required

A. Plating Human ES or IPS Cells for Transfection

The following instructions are for harvesting single human ES or iPS cells from a 6-well plate for transfection in 24-well plates. If using other cultureware, adjust volumes accordingly.

  1. Coat cultureware with Matrigel® and bring to room temperature (15 - 25°C) for at least 30 minutes prior to use.
  2. Warm (15 - 25°C) sufficient volumes of mTeSR™1, CloneR™, DMEM/F-12, and ACCUTASE™.
  3. Prepare Single-Cell Plating Medium by adding CloneR™ to mTeSR™1 at a 1 in 10 dilution.
    Example: To prepare 10 mL of Single-Cell Plating Medium, add 1 mL of CloneR™ to 9 mL of mTeSR™1.
  4. Aspirate matrix from coated cultureware and add 0.5 mL of Single-Cell Plating Medium to each well to be seeded.
  5. Use a microscope to visually identify regions of differentiation (if any) in the wells to be passaged. Mark these using a felt tip or lens marker on the bottom of the plate. Remove regions of differentiation by scraping with a pipette tip or by aspiration.
  6. Aspirate the remaining medium from the well and add 1 mL of ACCUTASE™. Incubate the plate at 37°C and 5% CO2 for approximately 5 minutes (incubation time may vary with different cell lines).
  7. Using a pipettor fitted with a 1000 μL tip, gently wash the cells from the surface of the plate by spraying the solution directly onto the colonies. Pipette the suspension up and down 2 - 3 times to break up small aggregates into single cells.
  8. Transfer the cell suspension to a 15 mL conical tube containing at least 5 mL DMEM/F-12 and mix by flicking the tube 2 - 3 times.
  9. Centrifuge the suspension at 300 x g for 5 minutes. Discard supernatant and resuspend cell pellet in 1 mL of Single-Cell Plating Medium.
  10. Count cells using a hemocytometer or automated cell counter.
  11. Add 1.25 x 105 cells per well to the plate prepared in step 4. Move the plate in several quick, short, back-and-forth, and side-to-side motions to evenly distribute cells.

    NOTE: Optimal cell density may vary with different cell lines and is dependent on when the cells will be harvested (typically 48 - 72 hours after plating).
  12. Incubate at 37°C and 5% CO2 for 24 hours. Refer to Figure 1 for a representative image of cell morphology following 24 hours of incubation.

Figure 4. Typical Cell Density of WLS-1C Human iPS Cell Line 24 Hours After Plating 1.25 x 105 Single Cells per Well

B. Preparation of ArciTect™ CRISPR-Cas9 RNP Complex for Chemical Transfection

Prepration of RNP and transfection should be performed approximately 24 hours after plating ES or iPS cells.

  1. Prepare 5 μM guide RNA by combining crRNA, tracrRNA, and Annealing Buffer in a microcentrifuge tube as indicated in Table 2. The volume below will provide sufficient reagent to transfect 4 wells of a 24-well plate; scale as needed. Mix thoroughly.
  2. In a thermocycler or heating block, incubate guide RNA mixture at 95°C for 5 minutes followed by 60°C for 1 minute. Cool to room temperature (15 - 25°C). If not used immediately, store at -80°C for up to 6 months.

  3. Table 7. Preparation of 5 μM Guide RNA

  4. Prepare a 5 μM Cas9 Nuclease solution in a microcentrifuge tube as shown in Table 3. This will provide sufficient reagent to transfect one well; if more transfections are required adjust volumes as needed. Mix thoroughly.
    NOTE: The amount of Cas9 to add will depend on the concentration and molecular weight of the Cas9 purchased, as shown in Table 3.

  5. Table 8. Preparation of 5 μM ArciTect™ Cas9 Nuclease Solution

  6. To prepare the RNP Complex Mix, combine components in a microcentrifuge tube as indicated in Table 4. Adjust component amounts according to the desired number of transfected wells. Mix thoroughly.

  7. Table 9. Preparation of 5 μM RNP Complex Mix for Chemical Transfection

    NOTE: If two or more guide RNAs are to be used in the experiment e.g. using ArciTect™ Cas9 Nickase, each RNP complex should be prepared separately.

    NOTE: May require optimization with different cell lines. A 1:2 (shown) to 1:4 molar ratio of Cas9 to guide RNA is recommended.

  8. Incubate the RNP Complex Mix at room temperature (15 - 25°C) for 10 minutes. While RNP complex forms, proceed immediately to section C.

C. Chemical Transfection of Human ES or iPS Cells with RNP Complex Using the TransIT-X2® Dynamic Delivery System

Preparation of RNP and transfection should be performed approximately 24 hours after plating ES/iPS cells.

  1. To prepare the Transfection Mix, combine components in a microcentrifuge tube as indicated in Table 5. Adjust component amounts according to the desired number of transfected wells. Mix thoroughly by pipetting up and down.

  2. Table 10. Preparation of Transfection Mix

  3. Incubate the Transfection Mix at room temperature (15 - 25°C) for 10 minutes.
  4. Remove the medium from cells plated onto 24-well plates the previous day, and replace with 450 μL of room temperature (15 - 25°C) mTeSR™1 per well. Place plate in a 37°C and 5% CO2 incubator.
  5. To prepare RNP Transfection Mix, combine components in a microcentrifuge tube in the order listed in Table 6. Volumes shown are for a single well; scale accordingly to accommodate all RNP complexes and replicates.

  6. Table 11. Preparation of RNP Transfection Mix

  7. Pipette the RNP Transfection Mix up and down to mix thoroughly.
  8. Incubate the RNP Transfection Mix at room temperature (15 - 25°C) for 20 minutes; do not exceed 30 minutes.
  9. Add 50 μL of RNP Transfection Mix dropwise per well of the 24-well plate prepared in step 3. Mix by gently moving the plate back and forth 2 - 3 times.
  10. Incubate the plate at 37°C and 5% CO2.
  11. Perform a full medium change every 24 hours with 500 μL of room temperature (15 - 25°C) mTeSR™1.
  12. If using Cas9-eGFP Nuclease, the transfection efficiency can be assessed 12 - 24 hours after transfection by flow cytometry (see Figure 2).
  13. Culture cells for 48 - 72 hours after transfection to allow genome editing to occur. Assess genome editing efficiency by T7 Endonuclease I Assay (see Figure 3). The cells can then be used to generate clonal ES or iPS cell lines and assess genome editing by T7 Endonuclease I assay and/or DNA sequencing. Refer to the CloneR™ Product Information Sheet (Document #DX21725) for instructions on subcloning ES or iPS cell lines.

Figure 5. Cas9-eGFP Detection by Flow Cytometry

WLS-1C (left) or STiPS-M001 (right) iPS cells were transfected with RNP complex containing Cas9-eGFP; eGFP was detected by flow cytometry 24 hours after transfection. Filled histogram: Non-transfected control; Solid line histogram: Cas9-eGFP-transfected cells.

Figure 6. INDEL Detection by T7 Endonuclease I Assay

H1 ES cells or WLS-1C iPS cells were edited using ArciTect™ Human HPRT Positive Control Kit, and INDEL formation was assessed using the T7 Endonuclease I Assay.
Control: Non-transfected cells; Test: HPRT-edited

References

  1. Heim R et al. (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci USA 91: 12501–4.
  2. Glaser A et al. (2016) GFP to BFP conversion: a versatile assay for the quantification of CRISPR/Cas9-mediated genome editing. Mol Ther Nucleic Acids 5: e334.
  3. Paquet D et al. (2016) Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533: 125–9.