Title:Effective Downregulation of BCR-ABL Tumorigenicity by RNA Targeted CRISPR-Cas13a
Volume: 21
Issue: 3
Author(s): Aditya Singh and Prateek Bhatia*
Affiliation:
- Pediatric Hematology-Oncology Unit, Department of Pediatrics, Advanced Pediatric Centre, Post Graduate Institute of Medical Education and Research, Chandigarh,India
Keywords:
CRISPR, oncology, molecular biology, hematology, RNA cleavage, BCR-ABL.
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 chromosomes 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.
Methods: 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 was 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 the 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 the way for further research
in this direction.
