CRISPR/Cas9 Genome Editing in Hematopoietic Cells (Jurkat and TF-1)

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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
 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)
 
Support Materials
Case Studies

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

CRISPR Jurkat, CRISPR Blood

 

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).

 CASESTUDY-TF1-PM-validation

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.

CASESTUDY-TF1-PM-figure2

Figure 2. The sequence chromatograph of a positive clone with homozygous point mutation. The CGG->CAG mutation was marked by the vertical line.  

CASESTUDY-TF1-PM-figure3

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.

 
References

Applied StemCell publications and citations:

CRISPR Technology:

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