Testimonial:

Implementing CRISPR-Cas9 Technology using Transfection Reagents from RJH Biosciences

Background

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a leading gene editing tools widely used today. The CRISPR-Cas9 system can effectively and accurately target DNA sequences with the use of an RNA scaffold, known as sgRNA [1]. The sgRNA and Cas9 enzyme can edit DNA by Non-Homologous End Joining (NHEJ), DNA cleavage followed by DNA repair by the host, or by Homologous Recombination (HR), DNA cleavage followed by gene insertion [1]. Other CRISPR systems also exist such as the CRISPR Cas13a system that cleaves RNA [2] and engineered Cas enzymes for modified applications or DNA targeting strategies [3,4].

The CRISPR technology is currently being used in several health-related applications including genotyping, diagnostics, and therapeutics [5]. This technology is promising for hematological diseases and has been used for developing therapeutic strategies to treat blood cancers [6]. As the CRISPR-Cas9 system is commonly deployed as a mRNA or DNA system, successful delivery into cells using a functional carrier is essential [7].

Transfection reagents developed by RJH Biosciences are highly effective at transfecting nucleic acids in a variety of cell types. Our optimization protocols and screening services aid researchers and clinicians to develop tailored transfection reagents to ensure effective delivery every time. The following research was performed by a collaborating company in which they compared several RJH Biosciences reagents for the delivery of CRISPR-Cas9 plasmids into mammalian cells.

Materials and Methods

Approach 1. Plasmids containing both the Cas9 enzyme and single sgRNAs targeting either WTAP or RunX were designed and delivered into the kidney fibroblast 293-T cells. RJH Biosciences transfection reagent Prime-Fect was used for delivery and LipofectamineTM 3000 (Lipo3000) was used to compare gene editing efficiency. The pDNA (200 ng) was mixed with each transfection reagent at 1:10 ratio (w/w). The transfection agent-pDNA mixtures were introduced into the cell culture that had been previously seeded in multi-well plates. NHEJ was confirmed by endonuclease assays. All experiments were performed in triplicate.

T7 Endonuclease Assays was performed by amplifying wild-type and the edited genomic DNA using PCR. The wild-type and edited DNA PCR products were mixed, denatured, and annealed together forming heteroduplexes between the two DNA species. T7 endonuclease I, an enzyme that recognizes and cleaves mismatched DNA, was added to the heteroduplexes as per manufacturing instructions. Multiple bands observed by gel electrophoresis indicated endonuclease cleavage and therefore editing of genomic DNA. The intensities of the full length and cleavage products were used to determine the relative CRISPR editing efficiency.

Approach 2. In a related approach, a commercially available kit, GeneArtâ„¢ CRISPR Nuclease Vector with CD4 Enrichment Kit (Thermo Fisher) was used to undertake CRISPR. The kit contains all the needed components for expression of functional CRISPR/Cas9 genome editing tools in mammalian cells with a CD4 reporter. The cells used was breast cancer MDA-MB-231 cells and transfection efficiency was tracked using anti-CD4 antibodies and bead-based purification. Transfection was undertaken using Lipofectamine2000TM or other custom made reagents from RJH Biosciences that is under development. Gene editing efficiency was based on percentage of bead-purified cells.

Results and Discussion

Approach 1. To assess CRISPR editing of WTAP or RunX, endonuclease assays were performed. Observed fragmented PCR products as compared to the control (Figure 1) indicates the occurrence of editing by the CRISPR-Cas9 system. Transfection using Lipo3000 and Prime-Fect result in NHEJ in both WTAP and RunX genes, indicating positive transfection of pDNA. The average efficiency of gene knockdown between Prime-Fect and Lipo3000 is summarized in Figure 1. Prime-Fect from RJH Biosciences shows comparable gene editing efficiency to that of Lipo3000. The occurrence of similar editing efficiency confirms the success of RJH Biosciences to deliver CRISPR-Cas9 containing pDNA, as compared to the leading transfection reagents on the market.

CRISPR-Cas9 pDNA delivery using RJH Biosciences Transfection Reagents

Figure 1. (Left) Endonuclease assays of WTAP and RunX gene editing using CRISPR-Cas9. Transfection of the cells was performed with Prime-Fect and Lipo3000. Multiple DNA bands indicate positive NHEJ. (Right) The relative editing efficiency with Lipo3000TM and Prime-Fect. The editing efficiency of WTAP is in blue and RunX in orange.

Approach 2. Using the commercial GeneArtâ„¢ kit and corresponding transfection reagents, successful gene editing was demonstrated in MDA-MB-231 cells (Figure 2). The transfection reagents used in this system are now under development and they have not been released for commercial use. They are available for specific projects upon contact with the RJH Biosciences.

Figure2_CRISPR_Cas9_transfection_Reagents
Figure 2. CRISPR-Cas9 genome editing efficiency using a commercial kit designed for CD4 editing. Transfection of the cells was performed with and Lipo2000 and 4 transfection reagents from RJH Biosciences that is currently under development. The relative editing efficiency with RJH reagents matched or exceeded the efficiency obtained with the leading lipofection reagent.

References

  1. Doudna, J and Charpentier, E. Science (2014) 346, 1258096.
  2. Zhang, J., and You, Y. Cancer Biology and Medicine. (2020) 17, 6-8.
  3. Xia, P. et al. ACS Synthetic Biology (2020) 9, 2162-2171.
  4. Walton, R. et al. Science (2020) 368, 290-296.
  5. Lee, S. et al. Trends in Molecular Medicine (2020) 26, 337-350.
  6. Antony, J. et al. Advances in Cell and Gene Therapy (2018) 1: e10.
  7. Wang, M. et al. Gene Therapy (2016) 34, 144-150.

| Data courtesy of a collaborating university lab and a biotech company (names withheld).

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