Pediatric Hematology-Oncology Unit, Department of Pediatrics, Advanced Pediatric Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
Curr Gene Ther. 2021;21(3):270-277. doi: 10.2174/1566523221666210217155233.
To induce BCR-ABL gene silencing using CRISPR Cas13a.
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.
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.
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.
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.
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.
使用 CRISPR Cas13a 诱导 BCR-ABL 基因沉默。
CML 是一种由 9 号和 22 号染色体之间的相互易位形成的 BCR-ABL 融合基因驱动的多能干细胞克隆性骨髓增生性疾病。伊马替尼等酪氨酸激酶抑制剂药物是治疗的主要方法，而对这些药物有抗药性且没有合适的干细胞供体的病例预后较差。然而，随着基因编辑技术的快速发展，大多数研究现在都集中在开发针对单基因疾病的翻译模型，以期实现永久性治愈。
探索 RNA 靶向 CRISPR-Cas13a 系统在 CML 细胞系 K562 中有效敲低 BCR-ABL 融合转录本的潜在应用。
针对嵌合 BCR-ABL 基因设计了 CRISPR Cas13a crRNA，并将该系统作为双质粒系统转染到 CML 细胞系 K562 中。通过评估依赖 BCR-ABL 基因表达的下游基因的表达水平来衡量效果。还使用下一代测序来确定 CRISPR 对基因的影响。
CRISPR 系统成功地将依赖于激活的 BCR-ABL 激酶信号的下游基因[pCRKL 和 pCRK]的表达降低了 4.3 倍。CRISPR 处理细胞的活力也显著降低了 373.83 倍[p 值=0.000891196]。时间依赖性动力学也突出了长达 8 周的显著体外抑制活性[p 值：0.025]。根据牛津 MinION 下一代测序仪的 cDNA 测序数据，CRISPR 处理的细胞显示出 62.37%疑似切割的读数。
这些初步结果突出了 RNA 靶向 CRISPR 在 CML 等血液恶性肿瘤中的极好的潜在应用，并应为这一方向的进一步研究铺平道路。
