• Cell Line Model Generation

CRISPR/Cas9 Cell Line Service - Primary Immune & Hematopoietic Cells (Primary T Cells, Jurkat and TF-1) Genome Editing

Struggling to Genetically Modify Primary & Other Hard-to-edit Cell Lines? We can Help! Primary, Immune and Hematopoietic cell lines are notoriously difficult to transfect and modify, but a crucial toolset for immuno-oncology research. ASC is the most experienced CRISPR/Cas9 genome engineering service provider in the industry with >1300 completed cell line projects under its belt, and has successfully modified several blood lineage cell lines with optimized, multi-approach design and strategies (see Technical Details for list of cell line modified):

  • Primary cells, adherent/ suspension cell lines; slow growing, hard-to-transfect
  • Variety of modifications: knockout, knock-in (point mutation, small/ fragment fragments)
  • Choice of deliverables: heterozygous/ homozygous clones
  • Isogenic cell lines generation
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Application Notes

Preclinical Models of Primary Immune Cells (T cells and Other Lymphocytes) for CAR-T and Immunotherapy Research!

Understanding the role of immune cells (T and B lymphocytes) in adaptive immunity, immuno-oncology and immune-related disorders is very crucial to developing successful immunotherapies. Primary immune cells (such as T and B lymphocytes) also provide a biorelevant in vitro model to understand immune system function and disorders, develop gene therapies/ immunotherapies (such as CAR-T), and drug target discovery and screening. However, genome editing in primary T lymphocyte and immune cell lines is extremely difficult and restrictive due to limited passaging potential, single cell clonability, very low efficiency for genome editing. But, ASC has > 10 years experience in CRISPR and genome editing technologies and our scientists are more than up to this challenge. As a long-standing genome engineering expert and pioneer CRISPR service provider, ASC has developed several techniques to engineer even these hard-to-edit cell lines. Using CRISPR-based non-viral transfection or lentiviral transduction protocols, we can design the best strategy to genetically manipulate your primary immune cell lines. We first evaluate the cell line for single cell culture conditions and to determine the best approach for your cell line of choice. We use various, well-optimized approaches for targeted genetic manipulation of your gene of interest: 

  • Transfection of non-viral CRISPR reagents: Plasmid-DNA expressed or in vitro transcribed (IVT) single guide RNA (gRNA); plasmid-DNA expressed Cas9 or Cas9-gRNA ribonucleoprotein (RNP) complex; and plasmid/ single stranded oligonucleotide (ssDNA) donor vectors - for knock-in projects
  • Transduction of lentiviral based reagents: Lenti-CRISPR or traditional lentiviral stable cell line generation techniques

Advantages of ASC’s Genome Editing Service

  • > 10 years of genome editing expertise
  • Well-optimized and proprietary protocols
  • Multi-approach genome editing strategies using complementary technologies: CRISPR/Cas9 and Lentivirus Stable Cell Line Generation and other technologies
  • Greater than 50% Cas9 cutting efficiency
  • Individualized protocols for each cell line’s unique properties – Not a one-size fits all approach!
  • Experience in generating >1300 cell lines in 200+ distinct mammalian cell lines, including hard-to-edit cell lines and primary cells
  • Variety of modifications: knockout, knock-in (small fragment/ point mutation)
  • Custom deliverables: footprint-free editing for clinical translation; choice of heterozygous/ homozygous mutations

Advantages of ASC’s Genome Editing Service

  • > 10 years of genome editing expertise
  • Well-optimized and proprietary protocols
  • Multi-approach genome editing strategies using complementary technologies: CRISPR/Cas9 and Lentivirus Stable Cell Line Generation and other technologies
  • High editing efficiency: optimized protocols for >50% Cas9 cutting efficiency
  • Individualized protocols for each cell line’s unique properties – Not a one-size fits all approach!
  • Experience in generating >1300 cell lines in 200+ distinct mammalian cell lines, including hard-to-edit cell lines and primary cells
  • Variety of modifications: knockout, knock-in (small fragment/ point mutation)
  • Custom deliverables: footprint-free editing for clinical translation; choice of heterozygous/ homozygous mutations

 Applications:

  • Biorelevant models to elucidate the mechanism and signal transduction of human/mammalian immune system
  • Disease modeling for immuno-oncology, immunodeficiency/ autoimmune and immune-related disorders
  • Feasibility studies for CAR-T and other immunotherapy research

Deliverables:

  • Two (2) vials of 2 x 10^5 cells/ vial of confirmed engineered cell line either as stable single cell clones (as determined by milestone 1 - evaluation of cell line for single cell clonability) or clonal pools (if single cell cloning is not feasible)
  • Detailed milestone and final report

Timeline: in as little as 4 months (varies by project type)


Examples of Gene Editing in Primary Immune T Cells

EXAMPLE 1: To generate a PD1 Knockout (PD1 KO) using CRISPR/Cas9 in primary human pan-T cells (include CD4 and CD8 T cells isolated from PBMCs)

appnote-crispr-geneediting-primary-tcell-pd1-knockout

Figure 1. Single guide RNAs (g1 and g2) were designed to guide Cas9 nuclease to cut at exon 2 of the Programmed Cell Death Protein 1; PD1 (PDCD1 (CD279) gene, a gene that plays a critical role (anti-tumor or tumorigenic) in several types of cancer.1 

appnote-crispr-geneediting-primary-tcells-pd1-ko-2

Figure 2. The two guide RNAs and Cas9 ribonucleoprotein complex (Cas9 RNP) were transfected into primary Pan-T cells (CD4+, CD8+ T cells) using two nucleofection programs (Prog 1 and 2). T7 Endonuclease assay to evaluate gRNA-Cas9 cutting efficiency showed desired cutting at the targeted locus. WT: wild type.

A. appnote-cripsr-geneediting-primary-tcell-pd1-ko-3    B. appnote-crispr-geneediting-primary-tcell-pd1-ko-4


Figure 3. A. PD1 KO T-cells quantification and B. CD4/CD8 ratio estimated by Flow Cytometry. A. Pooled population of PD1-KO T cells showed a reduced expression of PD1 at 3-4 days post-second activation with CD3/CD28 T cell activator complexes. Results shown includes data from N = 7 experiments. B. Representative results from one experiment showing CD4 and CD8 expression in wild type (WT) and PD1 KO T cells.

References:

1. Yao, H., Wang, H., Li, C., Fang, J. Y., & Xu, J. (2018). Cancer Cell-Intrinsic PD-1 and Implications in Combinatorial Immunotherapy. Frontiers in immunology, 9, 1774. doi:10.3389/fimmu.2018.01774


Technical Details

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."

- Universitat Pompeu Fabra

Below is a selected list of human and murine blood lineage cell lines we have modified:

Cell

Species

Tissue

Cell Type

Disease

BCWM-1*

Human

Bone marrow

Lymphoplasmacytic

Waldenstrom macroglobulinemia

FTC-133

Human

Thryoid

Thyrocytes

Follicular thyroid carcinoma

HMC1.2

Human

Peripheral blood

Mast cell

Mast cell leukemia

HUDEP-2

Human

Umbilical cord

Erythroid progenitor

Normal

Jurkat

Human

Peripheral blood

T lymphocyte

Acute T cell leukemia

Jurkat (Clone E6-1)

Human

Peripheral blood

T lymphocyte

Acute T cell leukemia

JVM2

Human

Peripheral blood

Lymphoblast

Mantle Cell Lymphoma

K562

Human

Bone Marrow

Lymphoblast

Chronic myelogenous leukemia (CML)

KG-1

Human

Bone

Lymphoblast

Acute myelogenous leukemia

KHYG-1*

Human

Peripheral blood

T lymphocyte

Natural killer cell leukemia

MWCL-1

Human

Bone marrow

Lymphoplasmacytic

Waldenstrom macroglobulinemia

T2

Human

Blood lineage

Lymphocyte

 

TF-1

human

Bone marrow

Erythroblast

Erythroleukemia

U937

Human

Lymphocyte

Monocyte

Histiocytic lymphoma

MOLM-13

Human

Peripheral blood

Monocyte-like

Acute myeloid leukemia

EML

Mouse

Bone marrow

Basophil

Normal

RAW 264.7

Mouse

Ascites

Macrophage

Abelson murine leukemia virus-induced tumor

Sp2/0-Ag14

Mouse

Spleen

B lymphocyte

Normal

CRISPR/Cas9 cell line service timeline and workflow:

 Service Time Deliverables
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)
Case Studies

You can find more case studies at:

1. Double Knockout in Jurkat Cells

2. Large Fusion Gene Knock-in in a Cancer Cell Line


Case Study 1: CRISPR/Cas9-mediated gene knock-ins in Jurkat cells

Case study 1 image - CRISPR cell line 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 TF-1 Cells

 

 gRNA validation graph image - CRISPR cell line gRNA validation

Figure 1. gRNA activity evaluation by deep sequencing. Two gRNAs candidates were selected according to the targeted region and off-target profile, and validated in K562 cells by Next Generation Sequencing (NGS). gRNA16 (g16) was chosen for subsequent gene editing.  


Figure 1 image - CRISPR Cell Line and Cas9 Gene Editing Figure 1

Figure 2. TF1 cells were transfected with the gRNA (g16) 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. The sequence chromatograph of a positive clone with homozygous point mutation. The CGG->CAG mutation was marked by the vertical line.  

Figure 2  image - CRISPR Cell Line and Cas9 Gene Editing Figure 2

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. Three homozygous positive clones and two heterozygous clones were obtained.  Further verification and off target analysis was performed for this case.

 
 
Publications
  • Jang, Y., Choi, J., Park, N., Kang, J., Kim, M., Kim, Y., & Ju, J. H. (2019). Development of immunocompatible pluripotent stem cells via CRISPR-based human leukocyte antigen engineering. Experimental & Molecular Medicine, 51(1), 3.
  • Colomar-Carando, N., Meseguer, A., Jutz, S., Herrera-Fernández, V., Olvera, A., Kiefer, K., ... & Vicente, R. (2018). Zip6 Transporter Is an Essential Component of the Lymphocyte Activation Machinery. The Journal of Immunology, ji1800689.
  • Tanic, J. (2018). A Role for Adseverin in the Invasion and Migration of MCF7 Breast Adenocarcinoma Cells (Doctoral dissertation).
  • Lizarraga, S. B., Maguire, A. M., Ma, L., van Dyck, L. I., Wu, Q., Nagda, D., ... & Cowen, M. H. (2018). Human neurons from Christianson syndrome iPSCs reveal allele-specific responses to rescue strategies. bioRxiv, 444232.
  • Tanaka, H., Kondo, K., Chen, X., Homma, H., Tagawa, K., Kerever, A., ... & Fujita, K. (2018). The intellectual disability gene PQBP1 rescues Alzheimer’s disease pathology. Molecular Psychiatry, 1.
  • Yin, Y., Garcia, M. R., Novak, A. J., Saunders, A. M., Ank, R. S., Nam, A. S., & Fisher, L. W. (2018). Surf4 (Erv29p) binds amino-terminal tripeptide motifs of soluble cargo proteins with different affinities, enabling prioritization of their exit from the endoplasmic reticulum. PLoS biology, 16(8), e2005140.
  • Selvan, N., George, S., Serajee, F. J., Shaw, M., Hobson, L., Kalscheuer, V. M., ... & Schwartz, C. E. (2018). O-GlcNAc transferase missense mutations linked to X-linked intellectual disability deregulate genes involved in cell fate determination and signaling. Journal of Biological Chemistry, jbc-RA118.
  • Smalley, E. (2018). FDA warns public of dangers of DIY gene therapy. https://doi.org/10.1038/nbt0218-119
  • Chai, S., Wan, X., Ramirez-Navarro, A., Tesar, P. J., Kaufman, E. S., Ficker, E., ... & Deschênes, I. (2018). Physiological genomics identifies genetic modifiers of long QT syndrome type 2 severity. The Journal of clinical investigation128(3).
  • Boi, S., Ferrell, M. E., Zhao, M., Hasenkrug, K. J., & Evans, L. H. (2018). Mouse APOBEC3 expression in NIH 3T3 cells mediates hypermutation of AKV murine leukemia virus. Virology518, 377-384. https://doi.org/10.1016/j.virol.2018.03.014.
  • Molinski, S. V., et al. (2017). Orkambi® and amplifier co‐therapy improves function from a rare CFTR mutation in gene‐edited cells and patient tissue. EMBO Molecular Medicine, e201607137.
  • Petrovic, P. B. (2017). Myosin Phosphatase Rho-interacting Protein Regulates DDR1-mediated Collagen Tractional Remodeling (Doctoral dissertation, University of Toronto (Canada)).
  • Peng, L., Zhang, H., Hao, Y., Xu, F., Yang, J., Zhang, R., ... & Chen, C. (2016). Reprogramming macrophage orientation by microRNA 146b targeting transcription factor IRF5. EBioMedicine14, 83-96.
  • Hu, J. K., Crampton, J. C., Locci, M., & Crotty, S. (2016). CRISPR-mediated Slamf1Δ/Δ Slamf5Δ/Δ Slamf6Δ/Δ triple gene disruption reveals NKT cell defects but not T follicular helper cell defects. PloS one11(5), e0156074.
  • Smalley, E. (2016). CRISPR mouse model boom, rat model renaissance. Nature Biotechnology. 34, 893–894.
  • Baker, M. (2014). Gene editing at CRISPR speed. Nature biotechnology32(4), 309-313.
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