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Successful CRISPR genome editing in hard-to-transfect cells (i.e., Jurkat cells)

Use of Alt-R® genome editing reagents and electroporation are key

Derived from T lymphocytic cells, the Jurkat cell line has been cloned into numerous sublines that are used in research laboratories to study immune system signaling, disease impact on the immune system, susceptibility of cancers to drugs and radiation treatment, and viral infection through chemokine receptors (such as with HIV). However, these cells are known for being notoriously difficult to transfect. This presents a problem for researchers interested in applying CRISPR genome editing technologies in these cells.

Lipofection protocols are often used for adherent, immortalized, eukaryotic cell lines, with protocol specifics often requiring optimization for different cell lines. For primary cells, non-dividing cells, and difficult-to-transfect cells, such as Jurkat cells, another delivery method, such as electroporation, is often required. The goal is to identify conditions that confer maximal editing efficiency and minimal cell toxicity.

We have already presented an optimized lipofection protocol for CRISPR reagents in HEK293 cells (see Alt-R® CRISPR-Cas9 System Users Guide). This article describes the optimization of electroporation conditions for delivery of CRISPR RNAs and Cas9 nuclease into Jurkat cells (Clone E6-1), using the Neon® Transfection System (Thermo Fisher Scientific). We also present data showing successful genome editing using our identified electroporation conditions for this cell line. The approach can be applied to other difficult-to-transfect cells, such as B cells, macrophages, and monocytes.


Reagent format

Scientists at IDT have established that CRISPR RNAs and Cas9 protein are most effectively delivered to transfected cell lines as a ribonucleoprotein (RNP) complex (see data in the article, Improve your genome editing with the Alt-R® S.p. Cas9 Nuclease 3NLS and modified crRNAs).

As part of the IDT Alt-R CRISPR-Cas9 System reagents, we offer crRNA, tracrRNAs, and the S.p. Cas9 Nuclease 3NLS. The RNAs are length optimized and chemically modified to further enhance genome editing by rendering the oligos less prone to degradation by nucleases. These modified RNAs thus have greater stability that leads to higher editing efficiencies, especially when used with the S.p. Cas9 Nuclease 3NLS.

For our Jurkat cell delivery optimization experiments, we therefore chose to use RNPs consisting of modified CRISPR RNAs complexed with Cas9 nuclease.


Electroporation optimization

For the optimization experiments, we designed an Alt-R® CRISPR-Cas9 System crRNA targeting the HPRT gene. The crRNA and tracrRNA were complexed in a 1:1 ratio with a final working concentration of 45 µM. We then generated RNP complexes by combining S.p. Cas9 Nuclease 3NLS protein and crRNA:tracrRNA in a 1:1.2 ratio, with a final working concentration of 18:21.6 µM.

The Jurkat cell line has been cloned into numerous sublines. For these experiments, we used Clone E6-1 Jurkat cells (ATCC® TIB-152™). 2 x 105 Jurkat cells were diluted in 10 µL Resuspension Buffer R (Neon Transfection System, Thermo Fisher Scientific) and added to 1 µL RNP complex. To test the effect of carrier DNA, 1 µL sequence optimized carrier DNA was added to a final concentration of 1.8 µM. For samples excluding carrier, we added 1 µL Buffer R.

Using a 10-µL Neon Transfection System tip, we followed the Neon® Optimization protocol (Publication Number MAN0001557, Revision A.0, page 22), which tests 24 electroporation settings, including voltage, pulse width, and number of pulses. Electroporated cells (10 µL) were mixed with 175 µL media, and 50 µL diluted cells were plated in triplicate in 100 µL pre-warmed media in a 96-well plate.

After 72 hours, we took images of the cell cultures to determine cell density. Genomic DNA was isolated to analyze genome editing using the T7EI mismatch endonuclease assay, as described in the Alt-R CRISPR-Cas9 System Users Guide. Note that the T7El assay underestimates total editing (see the sidebar, T7EI mismatch endonuclease assay for genome editing analysis).


T7EI mismatch endonuclease assay for genome editing analysis

We currently recommend using T7 endonuclease I (T7EI) instead of the Surveyor® endonuclease for CRISPR mutation detection. The T7EI method for genome editing analysis is simple and provides clean electrophoresis results. T7EI endonuclease is compatible with a broad range of PCR buffers and does not usually require purification of the PCR product prior to digestion. Note that T7EI activity is sensitive to the DNA:enzyme ratio, as well as incubation temperature and time [1]. T7EI is able to recognize insertions and deletions of ≥2 bases that are generated by NHEJ activity in CRISPR experiments [2]. Because T7EI does not recognize 1 bp indels, T7EI underrepresents the total editing. For a protocol, see the Alt-R® CRISPR-Cas9 System Users Manual.


Electroporation results

Table 1 lists the 24 electroporation test conditions. Cell density (reflective of cell toxicity) are scored using a 4 point scale; T7EI editing efficiency results are scored as percentages of detected edited alleles over non-edited alleles. Based on these two parameters, we identified optimal conditions for Neon electroporation of Jurkat cells as:

  • Electroporate with 3 pulses of 10 milliseconds, at 1600 volts

  • Include carrier DNA

Testing electroporation variables identifies delivery conditions compatible with successful genome editing in Jurkat cells

Table 1. Testing electroporation variables identifies delivery conditions compatible with successful genome editing in Jurkat cells. Test samples (n=24) were exposed to varying electroporation settings (voltage, pulse width, number of pulses) following the Neon® Optimization protocol (Publication Number MAN0001557, Revision A.0, page 22). (See text for experimental details.) Post electroporation, cells were cultured (72 hr) and then photographed to measure cell density. Genomic DNA was amplified and subjected to a T7El mismatch endonuclease assay to analyze genomic editing. Cell density results were scored using a 4 point scale; T7EI editing efficiency results were scored as percentages of detected edited alleles over non-edited alleles. Based on results for these two parameters, the best conditions for Neon electroporation of Jurkat cells were identified, noted by the checkmark (blue box) in test sample row Opt_24. For comparison, the conditions for electroporating Jurkat cells used by Liang et al (2015) [3] and provided by the Neon System optimization protocol for use with Jurkat cells [4] are also provided. Note that different Jurkat subclones may have been used in these previous reports. Also, Liang et al. (2015) electroporate less RNP than was used here.


Successful genome editing in electroporated Jurkat cells with carrier DNA augmenting editing efficiency

Figure 1. Successful genome editing in electroporated Jurkat cells with carrier DNA augmenting editing efficiency. Editing efficiency was plotted for the 24 electroporation conditions tested (Table 1), comparing absence vs presence of carrier DNA. In Table 1, we identified test sample Opt_24 as providing the best conditions for Neon electroporation of Jurkat cells, and have noted this data point here. For comparison, data from Liang X, et al. (2015) [3] and the Thermo Fisher Scientific website [4] were also plotted. Note that different Jurkat subclones may have been used in these previous reports. Also, Liang et al. (2015) electroporate less RNP than was used here.


Jurkat electroporation with CRISPR reagents leads to successful genome editing

The data presented here demonstrate that use of Alt-R® CRISPR-Cas9 System reagents for successful genome editing is not limited to easy-to-transfect cell types. With the resulting optimized electroporation conditions, we have shown Jurkat cell genome modification >75% + 0.4%. Keep in mind that RNA, protein, or RNP delivery conditions will require optimization for each individual cell line. The editing efficiency we obtained in this study is substantially higher than that achieved with the Thermo Fisher Scientific protocol, as described on their website [4], and is also higher than that described by Liang et al [3] (Figure 2). While these authors may have used distinct Jurkat subclones, which could explain some of the editing efficiency difference, our addition of a specific type of carrier DNA that enhances editing efficiency has clearly contributed to the superior data we obtained.

In cell types where lipofection has proven ineffective, electroporation should be considered as an alternative RNP delivery method. The electroporation optimization experiment presented here can provide a starting point for optimization of CRISPR reagent delivery in other cell types.


References

  1. Mean RJ, Pierides A, et al. (2004) Modification of the enzyme mismatch cleavage method using T7 endonuclease I and silver staining. Biotechniques, 36(5):758–760.

  2. Vouillot L, Thélie A, Pollet N. (2015) Comparison of T7EI and Surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3: Genes|Genomes|Genetics, 5(3):407–415.

  3. Liang X, Potter J, et al. (2015) Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol, 208:44–53.

  4. Invitrogen (2009) Neon® transfection system cell protocols: Jurkat microporation. [Online] Thermo Fisher Scientific. Available at www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/cell-culture/neon-protocols.par.97686.file.dat/jurkat-microporation.pdf [Accessed 15 June, 2016].


Product focus—genome editing with Alt-R® CRISPR Reagents

Alt-R CRISPR-Cas9 System

The Alt-R CRISPR-Cas9 System includes all the reagents needed for successful genome editing. Based on the natural S. pyogenes CRISPR-Cas9 system, the Alt-R CRISPR-Cas9 System offers numerous advantages over alternative methods:

  • Higher on-target potency than other CRISPR systems
  • Precision control with delivery of Cas9 ribonucleoprotein (RNP)
  • Efficient delivery of the RNP with lipofection or electroporation
  • No toxicity or innate immune response activation, in contrast to in vitro transcribed Cas9 mRNA and sgRNAs

Learn more about the Alt-R CRISPR-Cas9 System.


Alt-R CRISPR-Cpf1 System

The Alt-R CRISPR-Cpf1 System allows for new CRISPR target sites that are not available with the CRISPR-Cas9 System, and produces a staggered cut with a 5′ overhang. These reagents:

  • Enable genome editing in organisms with AT-rich genomes
  • Allow interrogation of additional genomic regions compared to Cas9
  • Require simply complexing the crRNA with the Cpf1 protein—no tracrRNA needed
  • Permit efficient delivery of the RNP into cells by electroporation

Learn more about the Alt-R CRISPR-Cpf1 System.


CRISPR support tools

Additional CRISPR reagents extend the ease-of-use and performance of the Alt-R system through options for enhanced RNP electroporation, fluorescent visualization, and genome editing detection.

Find out more about IDT’s entire line of CRISPR products.

Additional resources

CRISPR guide RNA format affects genome editing outcomes—Learn how use of different formats for the guide RNAs associated with CRISPR-Cas9 genome editing can lead to different editing outcomes. The optimized, short RNA oligos that make up the crRNA and tracrRNA components of the Alt-R® CRISPR-Cas9 System outperform other CRISPR guide RNA formats. Unlike DNA expression constructs, short RNA oligos are unable to incorporate into the target genome, resulting in cleaner editing.

6 pieces of data that will change how you set up your CRISPR-Cas9 experiments—Discover how you can improve the efficiency of CRISPR-Cas9 genome editing. IDT scientists evaluated several factors that influence how we design and perform genome editing experiments. Review the data and results for 6 important factors that were addressed. These experimental findings resulted in a set of potent CRISPR tools that are now offered as the Alt-R® CRISPR-Cas9 System.

Getting started with Alt-R® CRISPR-Cas9 genome editing—Webinar: Watch a recording of our webinar to learn about the components of the Alt-R CRISPR-Cas9 System, get information on designing Alt-R CRISPR crRNA oligos, and review the genome editing protocol from the user guide.


Author: Rolf Turk, PhD, is a staff scientist, and Ellen Prediger, PhD, is a senior scientific writer, both at IDT.

© 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.


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Product page:

Alt-R CRISPR-Cas9 System

Webinar:

New RNA tools for optimized CRISPR/Cas9 genome editing

Learn how research conducted at IDT led to the development of a potent new set of CRISPR-Cas9 genome editing tools.

Poster:

Quantitative Measurement of CRISPR/Cas9 Gene Editing at the Level of Genomic DNA for sgRNA Site Selection Algorithm Development

User guide:

Alt-R CRISPR-Cas9 System User Guide