Abstract
CRISPR/Cas genome editing is a powerful tool to precisely induce chromosomal breaks and to modify genes of interest. Cas9, an RNA-guided DNA endonuclease derived from Streptococcus pyogenes, is able to generate double stranded breaks (DSBs) in the genomic locus to where it is directed by its guide RNA (gRNA) component. The DSBs are subsequently repaired by one of the two main host repair mechanisms: the error-prone Non-homologous end joining (NHEJ) pathway or the very specific Homology-directed repair (HDR) pathway. We aimed to use CRISPR/Cas genome editing to generate the Fip1l1-Pdgfra and Nup214-Abl1 fusion genes by inducing chromosomal rearrangements in the interleukin-3 dependent Ba/F3 cell line.
Prior to generating the chromosomal rearrangements, we optimized CRISPR/Cas genome editing in Ba/F3 cells, by targeting Cas9 to exon 24 of CD45, a cell surface transmembrane protein, of which inactivation can be easily detected by flow cytometry. Electroporation of Ba/F3 cells with plasmids expressing Cas9 and the specific guide RNA led to efficient inactivation of the CD45 gene, as measured by flow cytometry (30% of the cells showed loss of CD45 expression). The use of the Cas9 nickase variant led to an increased efficiency of CD45 inactivation with 58% of the cells showing loss of CD45 expression. We then extended these studies to assess the efficiency of homology-directed repair to introduce a specific mutation, using a single strand donor template to generate a premature stop codon in exon 24 of CD45. The successful introduction of the novel stop codon in CD45 was confirmed by PCR amplification of the targeted exon followed by massive parallel sequencing (MiSeq, Illumina) and we observed this endogenous mutation in 80% of the Ba/F3 clones.
Having optimised the use and efficiency of CRISPR/Cas in Ba/F3 cells, we aimed to introduce double stranded breaks simultaneously in the genes Fip1l1 and Pdgfra to generate a cell based model for the FIP1L1-PDGFRA fusion gene as observed in chronic eosinophilic leukemia. Double strand breaks were introduced in Fip1l1 exon 23, 31, 32 or 34 together with simultaneous breaks in Pdgfra exon 12, both located on mouse chromosome 5. Upon IL3 removal, cells harbouring the deletion and fusion gene were able to survive, grow and form colonies in semi-solid medium, as was shown before for Ba/F3 cells transduced with retroviral vectors expressing FIP1L1-PDGFRA. The presence of the deletion was confirmed by PCR, and fusion protein expression was detected by Western blotting. A fusion between exon 1 of Fip1l1 and exon 12 of Pdgfra could also transform the cells, which confirmed earlier findings that the transforming capacities of the fusion protein are independent of Fip1l1 and dependent on the interruption of the juxtamembrane region of PDGFRA.
The expression and phosphorylation levels of Fip1l1-Pdgfra were compared between the CRISPR/Cas generated Ba/F3 cells and retrovirally transduced cells overexpressing FIP1L1-PDGFRA. As expected, retrovirally transduced cells showed a much higher protein expression level of FIP1L1-PDGFRA, and much stronger phosphorylation compared to the CRISPR/Cas generated cells, in which the endogenous Fip1l1 promoter is used to drive the expression of the fusion protein. We also observed a difference in sensitivity to inhibition by imatinib, a kinase inhibitor with strong activity against PDGFRA.
The same strategy was followed to generate a fusion between Nup214 and Abl1, as observed in a subset of T-cell acute lymphoblastic leukemia cases. Ba/F3 cells harbouring the Nup214-Abl1 fusion gene were able to survive and grow independent of IL3. The presence of the fusion gene was confirmed by PCR, and fusion protein expression was detected by Western blotting.
Taken together, these data show that CRISPR/Cas induced chromosomal translocations in cells more faithfully recapitulate gene expression levels and sensitivity to chemotherapeutics when compared to retroviral transduction based expression of an oncogene.
In conclusion, we have now designed and implemented an optimised platform to use CRISPR/Cas genome editing in Ba/F3 cells and measure gRNA efficacy by massive parallel sequencing. Our data confirm that the CRISPR/Cas genome editing system can be used to generate chromosomal rearrangements in Ba/F3 cells and provides a method to generate improved cell based models for the study of oncogenic tyrosine kinases.
Disclosures
No relevant conflicts of interest to declare.
Establishing CRISPR/Cas9 in Lipomyces starkeyi