CRISPR Cell Line Service - CRISPR/Cas9 Genome Editing in Hematopoietic Cells (Jurkat and TF-1)
CRISPR Cell Line Service:
"We contacted Applied StemCell for generating a CRISPR Knockout line attending at their expertise working with the Jurkat cell line. They were very professional and efficient generating the cell line in a very short time and the price was very competitive compared with other companies in the market. I recommend their services for other customers."
Applied StemCell is excited to announce that we can now genetically modify human blood lineage cell lines including Jurkat and TF-1 cells using our CRISPR/Cas9 gene editing technology. CRISPR/Cas9 technology is a powerful tool for gene editing. However, the efficiency of gene editing in many blood derived cell lines is extremely low. By optimizing the technology and conditions we now can successfully generate genetically modified a CRISPR cell line with this technology.
Jurkat cells are an immortalized line of human T lymphocyte cells that are used to study acute T cell leukemia, T cell signaling, and the expression of various chemokine receptors susceptible to viral entry, particularly HIV. The TF-1 cell line was established by T. Kitamura, et al. in October 1987 from a heparinized bone marrow aspiration sample derived from a 35 year old Japanese male with severe pancytopenia.
|CRISPR/Cas9 Cell Line Service Timeline|
|1. Targeting DNA Vector Creation||6-14 weeks||Biweekly updates throughout service|
|gRNA Design and Construction (2-4 gRNAs)||2 weeks|
|gRNA in vitro Functional Validation (2-4 gRNAs)||2-4 weeks|
|Donor DNA Construction (knock-in or point mutations)||2-4 weeks|
|2. Cell Culture, Transfection, Optimization||2-4 weeks|
|3. Cell Culture, Transfection/ Electroporation, Selection, Screening, and Clone Confirmation by PCR or Sequencing||7-10 weeks|
|4. Cell Expansion and Cryopreservation||1-2 weeks||Genetically Engineered Cell-Line, 2 vials (2x105 cells/vial)|
- Webinar: CRISPR/Cas9 gene editing in Blood-Derived Immune Cells
- POSTER, Tricon Conference 2016, San Francisco, CA (March 10-11, 2016).
Case Study 1: CRISPR/Cas9-mediated gene knock-ins in Jurkat cells
Figure. High efficiency gene knock-in using CRISPR/Cas9 in Jurkat cells: Out of 34 clones sequenced, 15 show the knock-in gene in the desired location. The clones were sequenced using 2 sets of primers for the 5' and 3' homology arms. The 5' junction PCR yielded a 2 kb fragment while the 3' junction PCR yielded a 2.2 kb fragment.
Case Study 2: CRISPR Cell Line. Point Mutation created in human hematopoietic cell line
Purpose of the study: To generate a point mutation in TF-1 cells using CRISPR/Cas9 technology.
gRNA validation: gRNAs candidates were selected according to the targeted region and off target profile. Two candidates were cloned and transfected into K562 cells and deep sequencing was performed to determine gRNA activity (Figure1).
Figure 1. gRNA activity evaluation by deep sequencing . We consider gRNAs with normalized NHEJ frequencies greater than or equal to 15% good candidates for cell line and animal model creation projects. gRNA g16 was chosen for downstream gene editing.
Gene editing with CRISPR/Cas9: TF1 cells were transfected with the gRNA plasmid, Cas9-puro plasmid and a single stranded DNA donor with the intended mutation. To avoid repeated cutting by Cas9, a few silent mutations were introduced in the donor. Cells were transiently selected with puromycin and single cell cloning was performed. Two weeks later, the clones were duplicated and genotyping was performed by PCR and Sanger sequencing. Both homozygous positive clones (Figure 3) and heterozygous clones (Figure 4) were obtained.
Figure 2. The sequence chromatograph of a positive clone with homozygous point mutation. The CGG->CAG mutation was marked by the vertical line.
Figure 3. The sequence chromatograph of a positive clone with heterozygous point mutation. The CGG->CAG mutation, marked by the vertical line, exists only in one allele, similar to the intended silent mutations.
Conclusion: Genetic knock-in of a point mutation into Pre-T suspension cells was achieved using CRISPR/Cas9 technology. Three homozygous positive clones and two heterozygous clones were obtained. Further verification and off target analysis was performed for this case.
Applied StemCell publications and citations:
- Smalley, E. (2016) CRISPR mouse model boom, rat model renaissance. Nature Biotechnology. 34: 893-894.
- Baker, Monya. (2014) Gene editing at CRISPR speed, Nature Biotechnology. 32: 309–312.
- Ran, FA, et al. (2013). Cell, 154(6), 1380-1389.
- Wang, H., et al. (2013). Cell, 153(4), 910–918. http://doi.org/10.1016/j.cell.2013.04.025
- Mali, P., et al. (2013). Nature Biotechnology, 31(9), 833–838. http://doi.org/10.1038/nbt.2675
- Ran, FA, et al. (2013) Nature Protocols, 2281-2308 doi:10.1038/nprot.2013.143
- Yang, H., et al. (2013). Cell, 154(6), 1370-1379.
- Cho, SW., et al. (2013). Nature biotechnology, 31(3), 230-232.
- Cheng, AW., et al. (2013). Cell Research, 23(10), 1163–1171.
- Cong, L., et al. (2013). Science (New York, N.Y.), 339(6121), 819–823.
- Jinek, M., et al. (2013). http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3557905/
- Gilbert, LA., et al. (2013). http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3770145/
- Hsu, PD., et al. (2013). Nature Biotechnology, 31(9), 827–832.
- Ramalingam, S., et al. (2013). http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3663103/
- Shen, B., et al. (2013). Cell Research, 23(5), 720–723.
- Fu, Y., et al. (2013). Nature Biotechnology, 31(9), 822–826.
- Pattanayak, V., et al. (2013). Nature biotechnology, 31(9), 839-843.
- Koyanagi-Aoi, M., et al. (2013). PNAS, 110(51), 20569–20574.
- Mali, P., et al. (2013). Nature Methods, 10(10), 957–963.
- Bikard, D., et al. (2013). Nucleic Acids Research, 41(15), 7429–7437.
- Yang, L., et al. (2013). Nucleic Acids Research, 41(19), 9049–9061. http://doi.org/10.1093/nar/gkt555
- Hou, Z., et al. (2013). PNAS, 110(39), 15644-15649.
- Qi, LS., et al. (2013). Cell, 152(5), 1173-1183.
- Pennisi, E. (2013). The CRISPR Craze. Science, 341(6148), 833-836.
- Shalem, O., et al. (2014). Science, 343(6166), 84-87.
- Carroll, D. (2013). Staying on target with CRISPR-Cas. Nature biotechnology,31(9), 807.
- Walsh, RM., & Hochedlinger, K. (2013). PNAS, 110(39), 15514-15515.
- Chen, F., et al. (2011). Nature methods, 8(9), 753-755.
- Sanjana, NE., et al. (2012) Nature protocols, 7(1), 171-192.
- Kim, H., et al. (2011). Surrogate reporters for enrichment of cells with nuclease-induced mutations. Nature methods, 8(11), 941-943.
- van Nierop, GP., et al. (2009). Nucleic acids research, 37(17), 5725-5736.
- Zhang, SC., et al. (2001). In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nature biotechnology, 19(12), 1129-1133.
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