Effective Downregulation of BCR-ABL Tumorigenicity by RNA Targeted CRISPR-Cas13a

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 chiCRISPR 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.meric 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.