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.

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 and can be sourced as either a two-part system where ArciTect™ crRNA and ArciTect™ tracrRNA are preannealed to form crRNA:tracrRNA duplexes prior to complexing with Cas9, or as a single guide RNA (sgRNA). Both custom sgRNA and custom crRNA can be designed using STEMCELL’s CRISPR Design Tool. Precise (knock-in) editing also requires the 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.

The following protocol provides tips for gRNA and donor template design, instructions for the preparation of CRISPR-Cas9 ribonucleoprotein (RNP) complexes, and their transfection into ES or iPS cells via electroporation and using the Mirus TransIT®-X2 System. We also provide recommendations for knock-in using a single-stranded oligodeoxynucleotide (ssODN) donor DNA template. In each protocol, the materials required are indicated on a per-well basis. These values will need to be scaled up for the actual number of wells in an experiment. It is recommended that each unique single guide RNA (sgRNA) or CRISPR RNA (crRNA) be tested in duplicate wells. Multiple sgRNAs or crRNAs are also often tested when a new gene is being targeted, as they will have different efficiencies at the target gene and at off-target sites. Optimization of conditions such as cell density or Cas9:guide RNA ratio may be desired for some difficult-to-transfect cell lines or refractory genomic locations.

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 (Catalog #85850) or (Catalog #05825), refer to the Technical Manual: Maintenance of Human Pluripotent Stem Cells in mTeSR™1 or mTeSR™ Plus.

This protocol uses (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.

Preparation of ArciTect™ sgRNA Working Solution or 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 crRNA and tracrRNA (Table 1) or 100 μM sgRNA (Table 2), as outlined below.



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



*200 μM is equal to 200 pmol/μL

Table 2. Resuspension Volume for 100 μM* ArciTect™ sgRNA



*100 μM is equal to 100 pmol/μL

3. Mix thoroughly. If not used immediately, aliquot and store at -20°C for up to 6 months or at -80°C for long-term storage. After thawing the aliquots, use immediately. Do not re-freeze.

Electroporation of Human ES and iPS Cells Using the Neon^® Transfection System and Lonza^® 4D-Nucleofector™ X Unit

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.

B. Annealing of crRNA:tracrRNA Duplexes

RNP complexes can be prepared using either two-part crRNA:tracrRNA duplexes, which require pre-annealing (see below), or sgRNA. If working with sgRNA, skip this section and continue to section C.

1. Prepare 80 μM crRNA:tracrRNA by combining crRNA, tracrRNA, and Annealing Buffer in a PCR tube as indicated in Table 3. Mix thoroughly.

Table 3. Preparation of 80 μM (80 pmol/μL) crRNA:tracrRNA duplexes


2. In a thermocycler or heating block, incubate 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 4. Adjust component volumes according to the desired number of transfections. Mix thoroughly.

Table 4. Preparation of RNP Complex Mix Using sgRNA or crRNA:tracrRNA



NOTE: May require optimization with different cell lines. A 1:2 to 1:8 molar ratio of Cas9 to gRNA is typically recommended.

2. Incubate the RNP Complex Mix at room temperature (15 - 25°C) for 10 - 20 minutes.
3. (Optional) Dilute 100 μM ssODN to 20 μM with Resuspension Buffer R (Neon^® Electroporation) or P3 Primary Cell Nucleofector™ Solution with Supplement 1 (4D-Nucleofector™ X Electroporation) as indicated in Table 5.

Table 5. Preparation of Diluted ssODN




4. After the RNP complex is formed, add either 2.5μl of Dilluted ssODN or appropriate buffer per electroporation device, to each RNP Complex Mix prepared in step 2 (Table 4), according to Table 6.

Table 6. Preparation of Complete RNP Complex Mix

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

Perform electroporation using either the Neon^® Transfection System (section a) or the Lonza^® 4D-Nucleofector™ X Unit (section b). This can be performed using the sgRNA RNP complex or the crRNA:tracrRNA RNP complex prepared in section D.

a) Electroporation Using Neon^® Transfection System

1. Aspirate supernatant from the cell pellet prepared in section C.
2. Resuspend 3 x 10⁵ cells in 7.5 μL of Resuspension Buffer R per electroporation condition and pipette up down to mix.
   
   Note:A range of 1 x 10⁵ to 1 x 10⁶ cells can be used per electroporation reaction.
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.



Table 7. Recommended Electroporation Conditions for Human ES or iPS Cells Using a Neon^® Transfection System



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

b) Electroporation Using Lonza^® 4D-Nucleofector™ X Unit

1. Aspirate supernatant from the cell pellet prepared in section C.
2. Resuspend 3 x 10⁵ cells in 17.5 μL of P3 Primary Cell Nucleofector™ Solution + Supplement 1 per electroporation condition and pipette up down to mix.
   
   Note: A range of 1 x 10⁵ to 1 x 10⁶ cells can be used per electroporation reaction.
3. Transfer 17.5 μL of the cell suspension to each 7.5 μL RNP Complex Mix (prepared in section D) and pipette up and down gently to mix, trying not to form air bubbles.
   
   Note: If air bubbles are present in the cuvette when the cells are electroporated, cell viability and transfection efficiency will be significantly reduced.
4. Transfer 20 μL of the cell suspension + RNP Complex Mix to one well of the 16-well Nucleocuvette™ Strip. Gently tap or use a pipette tip to ensure no air bubbles are present.
5. Set the Lonza^® 4D-Nucleofector™ X Unit to program code CA-137.
6. Place the Nucleocuvette™Strip in the Shuttle device of the 4D-Nucleofector™ X Unit, select OK to load the strip, and select Start to begin electroporation.

F. Post-Editing Culture

1. Immediately after electroporation, transfer cells to the warm (37°C) plate prepared in section A.
2. Mix by gently rocking the plate back and forth 2 - 3 times.
3. Incubate the plate at 37°C and 5% CO₂.
4. Perform a full medium change every 24 hours with 0.5 mL of room temperature (15 - 25°C) mTeSR™1 or mTeSR™ Plus.
5. Incubate the cells for 48 - 72 hours (or up to 7 days if confluency is low) after transfection for genome editing to occur.
6. Harvest cells for assessment of genome editing efficiency. Genomic DNA can be amplified by PCR using primers flanking the target region and (Catalog #76026), followed by sequencing of PCR products. Alternatively, (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.

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).

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

hPSCs were electroporated with RNP complexes containing GFP-targeting crRNA, co-delivered with ssODN encoding nucleotides for GFP to BFP conversion. Knockout and knock-in efficiency were measured 72 hours after electroporation. 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 ...[内容被截断]