• Cell and Gene Therapy Bioservice
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    Cell and Gene Therapy Bioservice

CRISPR/Cas9 Cell Line Service

No one-size fits all protocol in CRISPR Cell Line Genome Editing! ASC’s scientists understand culture behavior of different cell lines and use optimized protocols to engineer cell lines affordably, efficiently and in as fast as 2 months. To-date we have successfully harnessed the advantages of the CRISPR technology to engineer >1300 unique CRISPR cell line models with a variety of gene/ locus-specific modifications for applications in immuno-oncology, disease modeling, drug discovery/ screening, generating reference materials from engineered cell lines and more (see Technical Details section for a list of cell lines, types of modifications, and choice of deliverables). Add-on our downstream custom assay services for your cell line validation/ drug screening projects for a one-stop shop experience.

FAQs
What genome editing method do you use in your Cell Line service?
Is there a size limit on DNA to be inserted into the genome (CRISPR Knockin)?
Have you encountered difficulties in genome editing in cell lines?
How many guide RNAs do you typically design for a CRISPR Genome Editing Service?
Can you provide off-target analysis report?
Are you currently providing gene editing service to biotech/pharmatheutical companies as well?
Can we have a confidentiality disclosure agreement (CDA) before disclosing my project details?
What is the final deliverable product?
Products and Services
FAQs
What genome editing method do you use in your Cell Line service?
Is there a size limit on DNA to be inserted into the genome (CRISPR Knockin)?
Have you encountered difficulties in genome editing in cell lines?
How many guide RNAs do you typically design for a CRISPR Genome Editing Service?
Can you provide off-target analysis report?
Are you currently providing gene editing service to biotech/pharmatheutical companies as well?
Can we have a confidentiality disclosure agreement (CDA) before disclosing my project details?
What is the final deliverable product?
Technical Details

SCHEMATIC-CRISPR 

"Applied StemCell is noted in Nature Biotechnology as one of the select companies for CRISPR/Cas9 tools! Monya Baker, "Gene Editing at CRISPR Speed."

- Nature Biotechnology, 32: 309-312, April 2014


"Applied StemCell provided excellent cell line service from start to finish. I was very happy with the quality of their work.”

- University of Toronto

 

ASC uses CRISPR/Cas9 technology and proprietary algorithm for designing gRNAs which has been successful in genetically engineering >200 cancer cell lines, primary cells, induced pluripotent stem cells (iPSC) as well as in animal models. Using highly optimized and efficient protocols, we have engineered >1300 CRISPR cell lines to date for customers worldwide. As one of the first service providers for CRISPR cell line genome engineering, we understand CRISPR and cell lines. While it may be a powerful technology with a lot of promise, there are a lot of complexities that need to be taken into account to design a successful experiment. ASC’s cell line engineering scientists have several combined years’ of CRISPR/ Cas9 experience and know how to handle a variety of cell lines including hard-to-edit cell lines, primary cells, and stem cells for various types of genetic modifications and a >98% success rate in projects. We have optimized gRNA designing strategy that takes into account the modification that you need in your gene of interest and depending on the NHEJ or HDR-based repair mechanism to be used, off-target analysis and the cell line itself. We understand which cells are amenable for efficient single cell cloning and for which cells pooled clones are a better option; transfection conditions for various cell lines; and genotyping strategies to ensure the correct modifications in the right genomic locus. We offer customizable deliverables to engineer your CRISPR cell line such as: choice of homozygous or heterozygous clones; single cell/ pooled clones, point mutation without silent mutations, 100% footprint-free genome editing.

Let us workout the CRISPR engineering for you, so you can focus on your research. Starting from project design, we will work with you every step of the way to ensure you get your cell line that you want.

We offer precision custom engineering of Gene Fusion CRISPR Cell Lines to mimic clinically relevant cancer mutations in cell lines for a better understanding of cancer pathology and for developing successful therapies. In addition, we offer downstream cell-based assays to fully characterize and analyze the phenotype of your engineered CRISPR cell line.

Selected list of successfully modified mammalian cell lines# by ASC:

Cell

Species

Tissue

Cell Type

Disease

Blood Lineage Cells:    

BCWM-1* Human Bone marrow Lymphoplasmacytic Waldenstrom macroglobulinemia
EML Mouse Bone marrow Basophil Normal
FTC-133 Human Thryoid Thyrocytes Follicular thyroid carcinoma
HMC1.2 Human Peripheral blood Mast cell Mast cell leukemia
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 myelogenou leukemia
KHYG-1* Human Peripheral blood T lymphocyte Natural killer cell leukemia
MOLM-13  Human Peripheral blood Monocyte-like Acute myeloid leukemia
MWCL-1 Human Bone marrow Lymphoplasmacytic Waldenstrom macroglobulinemia
RAW 264.7 Mouse Ascites Macrophage Abelson murine leukemia virus-induced tumor
Sp2/0-Ag14 Mouse Spleen B lymphocyte Normal
T2 Human Blood lineage Lymphocyte  
TF-1  human Bone marrow Erythroblast Erythroleukemia
U937  Human Lymphocyte Monocyte Histiocytic lymphoma

Cancer Cell Lines:    

22RV1  Human Prostrate Epithelial Carcinoma
786-0 Human Kidney Epithelial Renal cell adenocarcinoma
A375   Human Skin Epithelial Malignant melanoma
A549 Human Lung Epithelial Carcinoma
AGS Human Stomach Epithelial Gastric adenocarcinoma
B16-F10 Mouse Skin Spindle/Epithelial-like Melanoma
CL-40  Human Colon Epithelial Colon carcinoma
CT-26 Mouse Colon Fibroblast Carcinoma
DLD-1 Human Colon Epithelial Dukes' type C, colorectal adenocarcinoma
H2030 Human Lung Epithelial Non-small cell lung cancer
H716 Human Cecum Epithelial Colorectal carcinoma
HAC15 Human Adrenal Epithelial-like Carcinoma
HBE  Human Lung Epithelial Lung cancer
HCT116 Human Colon Epithelial Colorectal carcinoma
HEK293 Human Embryonic kidney Epithelial  
HEK293T  Human Embryonic kidney Epithelial  
Hela  Human Cervix Epithelial Cervical cancer
HepG2 Human Liver Epithelial Hepatocellular carcinoma
HT1080 Human Coonective Tissue Epithelial Fibrosarcoma
HT29  Human Colon Epithelial Colorectal carcinoma
Huh7 Human Liver Epithelial Hepatocellular carcinoma
KYSE-270  Human Esophagus Epitheloid Esophageal squamous cell carcinoma
LNCaP  Human Prostrate Epithelial Prostrate adenocarcinoma
MALME-3M  Human Lung (metastatic) Fibroblast Malignant melanoma
Mc-38 Mouse Colon Epithelial Colon adenocarcinoma
MCF7  Human Mammary gland Epithelial Adenocarcinoma
mEERL Mouse Lung Epithelial Orpharyngeal squamous cell carcinoma
MKN1 Human Lymph node Epithelial Gastric adenosquamous carcinoma
Neuro-2a Mouse Brain Neuroblast Neuroblastoma
PANC1  Human Pancreas/duct Epithelial Epithelioid carcinoma
PC-3M Human Bone Epithelial Prostrate carcinoma
RCS Rat n/a Chondrocytes Chondrosarcoma
Renca Mouse Kidney Epithelial Renal adenocarcinoma
RKO Human Colon Epithelial Carcinoma
SBC-5  Human Lung n/a Small cell lung carcinoma
SCC-35 Human n/a Squamous cells Head and neck cancer
SH-SY5Y Human Bone Marrow Epithelial Neuroblastoma
SH-SY5Y (with eGFP) Human Bone Marrow Epithelial Neuroblastoma
T47D  Human Mammary gland Epithelial Ductal carcinoma
T84  Human Colon Epithelial Colorectal carcinoma
TC32 Human Bone n/a Neuroectodermal carcinoma
TOV-112D  Human Ovary Epithelial Primary adenocarcinoma
U-2 OS Human Bone Epithelial Osteosarcoma

Other Cell Lines:    

3617 Mouse Mammary gland Epithelial Normal
3T3-Swiss albino Mouse Embryo Fibroblast Normal
4T1* Mouse Mammary gland Epithelial Normal
AGMK GL37 African Green Monkey Kidney Epithelial Normal
ARPE-19 Human Eye Retinal pigmented Epithelium Normal
BEAS-2B Human Lung Epithelial Normal
BJ-hTERT Human Skin (foreskin) Fibroblast Normal
CHO-S Hamster Ovary Epithelial-like  
cTEC C9() Mouse Thymus Epithelial cTEC
Fibroblast (primary) Human   Primary fibroblast Normal
HaCaT Human Skin Keratinocyte Normal
HMEC Human Dermal endotheliym Dermal microvascular endothelium  
hTERT RPE Human Retina (pigmented epithelium) Epithelial    
IDH4  Human Lung Fibroblast IDH
MCF10  Human Mammary gland Epithelial Fibrocystic disease
NIH/3T3 Mouse Embryo Fibroblast Normal
PCCL3 Rat Thyroid Epithelium  
Podocyte Human Kidney Primary  
SW10 Mouse Neuronal Schwann cell Neuronal  

Stem Cells (iPSC/ ESC):    

Induced pluripotent stem cells (iPSC) Human PBMC/ Skin/ Cord blood PBMC/Fibroblasts Normal/ Disease
Embryonic stem cells (ESC) Human inner cell mass Embryonic stem cell Normal/ Disease
iPSC Mouse, Primate, Others Skin Fibroblast Normal
ESC Mouse, Rat, Macaque inner cell mass Embryonic stem cell Normal
Don’t see a particular model you are interested in? Contact us to learn about the full scope of our expertise and get a cell line model engineered precisely to your project requirements.

Applications: 

 

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)

We can use either transfected plasmids or oligos to create your cell line. For difficult-to-transfect cell lines, we use electroporation to introduce the gene modification. For Knock-in cell lines, the donor DNA contains either a fluorescent marker (such as GFP) or an antibiotic resistance gene (such as puromycin) for selection.

Case Studies

Click for Fusion Gene Knock-in Case Study

 Knock-out Case Study #1. Multi-gene knock-outs in human cancer cells.

Surveyor assays and sequencing results showed that a stable homozygous mutant cell line with a two base pair deletion in clone X was successfully produced, causing a frame shift in gene A.
Knock-out case study #1 - CRISPR points mutations- Knock-outs in human cancer cells
Sequence Data: Final clone X (homozygous) and WT, both forward and reverse sequence of Gene A.

Knock-out case study #1 Sequencing Data - CRISPR points mutations- Knock-outs in human cancer cells reverse sequence
Raw sequence data: Data shows reverse sequence results. Black box highlights the two base pairs present in the WT which are absent in clone X.


Knock-out Case Study #2: Precise deletion of a specific fragment from the target gene in a cancer cell line.

Two gRNA were designed to delete a specific fragment. Both homozygous and heterozygous clones were identified after screening and sequence verification. This example shows that CRISPR/Cas9 can be used for precise deletion.
 
 Knock-out case study #2 - CRISPR points mutations- knock-out case study 2

Knock-in Case Study #3: Point mutation knock-in in mouse fibroblasts.

A Cas9/gRNA-expressing vector was used to introduce a cut at the target site. A single-stranded oligonucleotide (ssODN) was used as a donor for homology-directed repair (HDR) at the cut site introduced by Cas9. Sequence data indicate a two nucleotide change of “CC” to “AG”.

Knock-in Case study #3 WT - CRISPR points mutations- Knock-in Case study 3 WT  Knock-in Case study #3 Mutant - CRISPR points mutations- Knock-in Case study 3 Mutant
               WT                            Mutant

 

Delivered Cell Line Engineering Projects (selected)
DLD-1 (Colon Cancer) Human
HCT116 (Colon Cancer) Human
HT29 (Colon Cancer) Human
RKO (Colon cancer) Human
HT1080 (Fibrosarcoma) Human
HeLa (Cervical cancer) Human
Breast Cancer Cells Mouse
HEK293 (Kidney) Human
B Lymphocyte Human
Erythroblast Human
iPSC  (various from disease/healthy) Human
HaCaT (Immortal Keratinocyte) Human
Immortalized Bronchial Epithelial Cell Line Human
Immortalized Fibroblast Puromycin Resistant Line Human
Huh7 (Liver) Human
Mammary Gland Epithelial Human
MEF Mouse
Glioblastoma Human
Immortalized Cementoblast  Mouse
Immortalized Hepatocytes Rat
Follicular thyroid cell line Rat
CHO (Hamster Ovary) Chinese Hamster
 
Publications

Applied StemCell publications and citations:

  • Panda, D., Gjinaj, E., Bachu, M., Squire, E., Novatt, H., Ozato, K., & Rabin, R. L. (2019). IRF1 maintains optimal constitutive expression of antiviral genes and regulates the early antiviral response. Frontiers in immunology10, 1019.
  • Pisapia, P., Malapelle, U., Roma, G., Saddar, S., Zheng, Q., Pepe, F., ... & Nikiforov, Y. E. (2019). Consistency and reproducibility of next‐generation sequencing in cytopathology: A second worldwide ring trial study on improved cytological molecular reference specimens. Cancer cytopathology127(5), 285-296.
  • Ilic, D. (2019). Latest developments in the field of stem cell research and regenerative medicine compiled from publicly available information and press releases from nonacademic institutions in October 2018. Regenerative medicine, 14(2), 85-92.
  • Simkin, D., Searl, T. J., Piyevsky, B. N., Forrest, M., Williams, L. A., Joshi, V., ... & Penzes, P. (2019). Impaired M-current in KCNQ2 Encephalopathy Evokes Dyshomeostatic Modulation of Excitability. bioRxiv, 538371. https://doi.org/10.1101/538371
  • 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.
  • (12), 1129-1133.
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