Genome Engineering: The CRISPR/Cas Revolution (2015) Cold Spring Harbor, NY, USA

by S. Kumar, X. Liang, X. Yu, H. Barbour, Y. Zou, J. Potter, J. Chesnut, and N. Ravinder; Thermo Fisher Scientific, Carlsbad, CA, USA - 09/24/2015

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Abstract

Genome editing in induced pluripotent stem cells (iPSCs) has been demonstrated to be highly effective for generating disease models for both monogenic and complex genetic disorders. For successful genome editing of iPSCs many factors need to be considered such as choice of growth media, genome editing tools, and nucleic acid (NA) delivery methods. Currently many genome editing tools require large quantities of NA, involve tedious cloning steps and are relatively more toxic to iPSCs. To overcome these challenges we developed a highly purified transfection grade CRISPR/Cas9 ribonucleoprotein (RNP) complex, which is highly efficient in genome editing with minimal toxicity. We also optimized a transfection condition to maximize delivery and genome editing in stem cells using CRISPR/Cas9 RNP complex. Discussed here are the results from different CRISPR/Cas9 formats tested across wide variety of cell types including stem cells. Using these formats we have edited mouse embryonic stem cells (ESCs) and human iPSCs with 80% to 60% genomic cleavage efficiencies, respectively. The methods described here facilitate efficient disease model generation thereby accelerating research in the field of gene therapy and regenerative medicine.

Introduction

Genome editing in induced pluripotent stem cells (iPSCs) has been demonstrated to be highly effective for generating disease models for both monogenic and complex genetic disorders. For successful genome editing and downstream application of iPSCs, many factors need to be considered, such as choice of growth media, extracellular matrix, genome editing tools, and nucleic acid (NA) delivery methods. Here we have described feederfree culture of stem cells and a genome editing protocol that can facilitate efficient disease model generation.

The clustered regularly interspaced short palindromic repeat (CRISPR) system from Streptococcus pyogenes has become a powerful technology for genome editing, and it can be used to rapidly generate engineered cell lines and model organisms. It is a simple system comprising a catalytic unit, Cas9, and a short noncoding guide RNA (gRNA) that confers target specificity. It is an attractive tool for large-scale genome engineering in a wide variety of hosts (1-3). We have developed various CRISPR-Cas9 formats that can be used to edit genomes in a wide variety of cell types, including stem cells (4). Using these formats we have achieved greater than 50% target-specific DNA cleavage in mouse embryonic stem cells (ESCs) and human iPSCs and ESCs (4). The methods described here show great potential as highly efficient gene editing tools in stem cells. 

Materials and methods

1. Mouse ESCs culture and transfection

Mouse E14Tg2a.4 ESCs were cultured on mouse inactivated embryonic fibroblasts (MEFs; strain ICR) in the presence of recombinant human leukemia inhibitory factor (LIF) in mouse ESC medium. Before transfection, cells were adapted to feeder-free conditions and maintained on plates coated with attachment factor protein in mouse ESC–conditioned medium.

Transfection: Lipid-based delivery of CRISPR components (option 1).

DNA format: 750 ng of All-in-one OFP plasmid DNA was transfected following Lipofectamine® 3000 Transfection Reagent user manual.

RNA format: Approximately 200 ng of in vitro–transcribed gRNA and 1 μg of GeneArt® CRISPR Nuclease mRNA were used, 2.5 μL of Lipofectamine™ MessengerMAX™ per transfection.

Protein format: For Cas9 RNP transfection, 125 ng of gRNA and 500ng of Cas9 protein were mixed and transfected using Lipofectamine™ RNAiMAX Electroporation based delivery of CRISPR components (option 2) 1X105 cells were resuspended in 10μl of Neon™ R-buffer which was precomplexed with and 200 ng of in vitro transcribed guide RNA and 1μg of Cas9 Nuclease. Best Neon electroporation conditions for RNPs: 1500V, 10ms, 3 pulse (Neon™ optimization no 23).

2. Human ESCs and iPSCs transfection

Option 1: Feeder-free adaptation of iPSCs for transfection Feeder-dependent human episomal Gibco iPSC line cultured on MEF feeder cells (strain ICR) in human ESC medium containing 20% KnockOut™Serum Replacement, 10 μM MEM Non-Essential Amino Acids Solution, 55 μM 2-mercaptoethanol, and 4 ng/mL human bFGF recombinant protein in DMEM/F12. Cells were adapted to feeder-free conditions before electroporation and transfection

Option 2: Culturing cells in feeder-free conditions in Essential 8™ Medium Cells harvested using TrypLE™ Express Enzyme by incubating for 2–3 minutes in a 37°C humidified incubator. Single-cell suspensions were prepared. For lipid-based transfection, a 24-well tissue culture plate coated with Geltrex® matrix was used. Each well was seeded with approximately 0.5 x 105 cells in 500μL of Essential 8™ Medium containing 10 μM ROCK inhibitor and allowed to recover overnight.

3. GeneArt CRISPR Cas9 Formats

GeneArt CRISPR Cas9 Formats

DNA: all-in-one plasmid

  • Reporter-based enrichment
  • Random integration concern
  • Need cell-specific promoter
  • Relatively more off-target events

mRNA: Cas9 mRNA

  • No footprint left behind
  • No promoter constraint
  • Ready-to-use
  • Controlled dosage
  • Fast turnover

Protein: Cas9 protein (RNP)

  • No footprint left behind
  • No promoter constraint
  • Ready to act
  • Controlled dosage
  • Fast turnover
  • Stable RNP complex

Results

Editing efficiencies in mouse ESCs

Figure 1. Editing efficiencies in mouse ESCs.
(A) Transfection of various formats of CRISPR/Cas9. 
Transfection of GeneArt™ CRISPR Nuclease Vector with OFP reporter (DNA), GeneArt™ CRISPR Nuclease mRNA (RNA), and GeneArt™ Platinum™ Cas9 Nuclease (Protein) in mouse ESCs. DNA transfection was performed using Lipofectamine™ 3000, Cas9 mRNA with Lipofectamine™ MessengerMAX reagent, and Cas9 Nuclease with Lipofectamine™ RNAiMAX. Cells were assayed for genomic cleavage of Rosa26 locus, 48 hours post transfection.
(B) Electroporation of various formats of CRISPR/Cas9. Various CRISPR/Cas9 formats (DNA, mRNA and Protein) were electroporated into mouse ESCs using 10μl Neon™ tips. 24 different Neon™ optimization conditions were tested and genomic cleavage assay for Rosa26 locus was performed 48 hours post electroporation.


TOP

Editing efficiency in human iPSCs (Feeder vs. Feeder free)

BOTTOM

iPSCs grown on Geltrex

Figure 2. Editing efficiency in human iPSCs (Feeder vs. Feeder free).

TOP: 
iPSCs grown on feeder cells 
(A) Transfection of Various CRISPR/Cas9 formats (DNA and mRNA) in Gibco™ human iPSCs grown on feeder layer. Before transfection, iPSCs were transferred to Geltrex™ matrix–coated plates in conditioned medium. Plasmid DNA and mRNA were transfected using Lipofectamine™ 3000 and Lipofectamine™ MessengerMAX transfection reagents, respectively. Genomic cleavage assay for HPRT locus was performed 72 hours post transfection. 
(B) Electroporation of Various CRISPR/Cas9 formats (DNA, mRNA and Protein) in Gibco iPSCs gown in conditioned medium. Single cells suspension was collected and 100K cells were electoporated using various CRISPR formats and genomic cleavage assay performed 72 hours post transfection.

BOTTOM: iPSCs grown on Geltrex™ matrix. Gibco iPSCs were grown on Geltrex coated plates in Essential 8™ Medium and transfected with CRISPR/Cas9 formats (mRNA and RNPs) using Lipofectamine™ MessengerMAX and RNAiMAX, respectively.
(A) Cell density before and 48 post transfection. Transfections were performed in presence of ROCK inhibitor and medium was changed 6 hours post transfection.
(B) Effect of cell detachment factors (Accuatse™ vs. TrypLE™ on genomic cleavage efficiency. Cells were transfected using various amount of gRNA (125-250ng) and Cas9 protein (500- 1000ng) and genomic cleavage assay performed 48 hours post transfection.
(C) Effect of medium change before and after transfection. Blue bar shows medium changes prior to transfection; red bar shows medium containing ROCK inhibitor at the time of transfection.
(D) Comparison of cleavage efficiency between CRISPR/Cas9 formats (mRNA and RNPs). Cells were transfected in duplicate with gRNA against HPRT locus and Cas9 (mRNA or Protein). Genomic cleavage assay was performed 48 hours post transfection.

Optimization of RNPs electroporation in human iPSCs/H9

Figure 3. Optimization of RNPs electroporation in human iPSCs/H9.
(A) Gibco iPSCs were grown on Geltrex™ in Essential 8 medium. Single cell suspensions were collected using TrpLE™ and 100 K cells were electroporated using 10 μl Neon™ tips. 200 ng of IVT gRNA against HPRT locus and 1.5 μg of Cas9 protein were complexed for 10 min at room temperature. Electroporation was performed using Neon electroporation system in R buffer. Preset 24 different Neon optimization conditions were tested and genomic cleavage assays were performed 72 hours post transfection.
(B) Electroporation of RNPs in Gibco iPSCs. 100k cells were electroporated using Neon electroporation condition- 1200V, 20 ms, 2 pulse.
(C) Electroporation of RNPs in human H9 cells. 100k cells were electroporated using Neon electroporation condition- 1200V, 20 ms, 2 pulse.


Cell viability and pluripotency of iPSCs post electroporation

Figure 4. Cell viability and pluripotency of iPSCs post electroporation.
(A) Single cell suspension of Gibco iPSCs were collected and electroporated using Neon electroporation system with various CRISPR/Cas9 formats (mRNA and RNPs). Three days post electroporation cells were stained using fluorescently labeled antibodies against pluripotency markers, and analyzed using flow cytometer.
(B) Various CRISPR/Cas9 formats (All-in-plasmid, mRNA and RNPs) were electroporated (best condition) in Gibco iPSCs and AP stained using 3 days post electroporation. 750 ng of All-in-one plasmid; 200ng of IVT and 500 ng of Cas9 mRNA; 200 ng of IVT and 500 ng of Platinum Cas9 nuclease.


Efficient creation of SNP in iPSCs using ssODN and CRISPR
Figure 5. Efficient creation of SNP in iPSCs using ssODN and CRISPR.
(A) CRISPR guide RNA design and ssODN for SNP change.
(B) Gibco iPSCs were transfected with CRISPR/Cas9 (mRNA) and 10 pico moles of ssODN (modified and unmodified ssODN), cleavage efficiency determined 72 hours post transfection.
(C) SNP efficiency were calculated by Sanger and Ion PGM sequencing.

Conclusions

  • Optimized transfection/electroporation conditions in mouse ESCs and human iPSCs/ESCs using CRISPR/Cas9 (mRNA and RNPs) that resulted in above 50-70 % genome editing efficiencies
  • Singularization of iPSCs with TrypLE™ showed higher editing efficiencies compared to cells collected with Accutase™
  • iPSCs transfected in presence of ROCK inhibitor showed higher genome editing efficiencies
  • iPSCs transfected with RNPs showed highest cell survivability
  • Highest HR efficiency for SNP were obtained using CRSIPR/Cas9 (mRNA) and ssODN
     

Acknowledgements

Rene Quintanilla, Mahalakshmi Sridharan, and Xin Yu