BIRC5 Gene Disruption via CRISPR/Cas9n Platform Suppress Acute Myelocytic Leukemia Progression

Abstract

Background: Acute myelocytic leukemia (AML) is a clonal malignancy resulting from the accumulation of genetic abnormalities in the cells. Human baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), encodes survivin, is one of only a handful of genes that is differentially over-expressed in numerous malignant diseases including AML.

Methods: The BIRC5 was silenced permanently in two AML cell lines, HL‑60 and KG-1, via the CRISPR/Cas9n system. After transfection of CRISPR constructs, genomic DNA was extracted and amplified to assess mutation detection. To evaluate BIRC5 gene expression, quantitative real-time PCR was performed. Also, MTT cell viability and Annexin‑V/propidium iodide flowcytometric staining were performed, and the data were analyzed using the Kolmogorov-Smirnov, Levene's, and ANOVA tests.

Results: The results indicated that Cas9n and its sgRNAs successfully triggered site-specific cleavage and mutation in the BIRC5 gene locus. Moreover, suppression of BIRC5 resulted in the reduction of cell viability, and induction of apoptosis and necrosis in HL60 and KG1 suggested that the permanent suppression of BIRC5 remarkably dropped the gene expression and cells viability.

Conclusion: This study reinforces the idea that BIRC5 disruption via Cas9n:sgRNAs has favorable effects on the AML clinical outcome. It thereby can be a promising candidate in a variety of leukemia treatments.

Keywords: Acute myelocytic leukemia; CRISPR; Gene editing; Survivin.

Fig. 1

Illustration of the designed CRISPR/Cas9n system for BIRC5 gene knockout. (A) Schematic diagram of BIRC5 gene exons and their corresponding nucleotide positions. (B) Presentation of sgRNA sequences-directed CRISPR/Cas9n system and their corresponding regions in BIRC5. Protospacer adjacent motif sequences are labeled in yellow; sgRNAs in brown, as well as sense and anti-sense strands of the BIRC5 gene are shown in navy blue and dark green, respectively

Fig. 2

CRISPR/Cas9n-mediated cleavage at BIRC5 locus in acute myelocytic leukemia cells. (A) PCR detection;(B) Surveyor assay of CRISPR/Cas9n activity in KG-1 and HL-60 cell lines. The numbers on the left represent the sizes of the DNA Ladder (bp). The numbers at the bottom of the gel indicate mutation percentages measured by band intensities

Fig. 3

Quantitative evaluation of BIRC5 expression in HL-60 (A) and KG-1 (B) cell lines via qRT-PCR 48 h after transfection. Relative expression values were normalized assigning the value of the cells in control groups to 1.0. Error bars represent mean ± SD of biological replicates from one experiment (p < 0.0001)

Fig. 4

Proliferation of the various groups-transfected HL-60 (A) and KG-1 (B) cells quantified using a MTT assay 48 h after transfection. The viability of the untreated cells was considered as 100%, and the viability of other groups is presented as the percentage of the untreated cells. Data were mean ± SD of three independent experiments (p < 0.0001)

Fig. 5

Targeting of survivin resulted in the induction of apoptosis in leukemic cell lines. Significantly enhanced apoptosis was monitored by flow cytometry in the treated versus untreated groups in HL-60 and KG-1 cell lines 48 h after transfection. (A) unstained, (B) control, (C), scramble, and (D) Cas9n. Representative cytofluorometric graphs are shown in (E) for HL-60 and KG-1 cell lines