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Effective downregulation of BCR-ABL tumorigenicity by RNA targeted CRISPR-Cas13a

Author(s):

Aditya Singh and Prateek Bhatia*  

Abstract:


Aim: To induce BCR-ABL gene silencing using CRISPR Cas13a.

Background: CML is a clonal myeloproliferative disorder of pluripotent stem cells driven by a reciprocal translocation between chromosome 9 and 22, forming a BCR-ABL fusion gene. Tyrosine-kinase inhibitor drugs like imatinib are the mainstay of treatment and cases resistant to these drugs have a poor prognosis in the absence of a compatible stem-cell donor. However, with rapid advancements in gene-editing technologies, most studies are now focusing on developing a translational model targeting single-gene disorders with a prospective permanent cure.

Objective: To explore the potential application of the RNA targeting CRISPR-Cas13a system for effective knockdown of BCR-ABL fusion transcript in a CML cell line, K562.

Method: CRISPR Cas13a crRNA was designed specific to the chimeric BCR-ABL gene and the system was transfected as a two-plasmid system into a CML cell line, K562. The effects were enumerated by evaluating the expression levels of downstream genes dependent on the expression of the BCR-ABL gene. Also, next-generation sequencing was used to ascertain the effects of CRISPR on the gene.

Results: The CRISPR system was successfully able to lower the expression of downstream genes [pCRKL and pCRK] dependent on the activated BCR-ABL kinase signal by up-to 4.3 folds. The viability of the CRISPR treated cells were also significantly lowered by 373.83-fold [p-value= 0.000891196]. The time-dependent kinetics also highlighted the significant in-vitro suppressive activity to last up to 8 weeks [p-value: 0.025]. As per the cDNA sequencing data from Oxford MinION next-generation sequencer, the CRISPR treated cells show 62.37% suspected cleaved reads.

Conclusion: These preliminary results highlight an excellent potential application of RNA targeting CRISPRs in Haematological neoplasms like CML and should pave way for further research in this direction.

Keywords:

CRISPR, oncology, molecular biology, hematology, RNA cleavage

Affiliation:

Pediatric Hematology-Oncology Unit, Department of Pediatrics, Advanced Pediatric Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, Pediatric Hematology-Oncology Unit, Department of Pediatrics, Advanced Pediatric Centre, Post Graduate Institute of Medical Education and Research, Chandigarh



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