Diagnostic applications and therapeutic option of Cascade CRISPR/Cas in the modulation of miRNA in diverse cancers: promises and obstacles

Abstract

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology is a molecular tool specific to sequences for engineering genomes. Among diverse clusters of Cas proteins, the class 2/type II CRISPR/Cas9 system, despite several challenges, such as off-target effects, editing efficiency, and efficient delivery, has shown great promise for driver gene mutation discovery, high-throughput gene screening, epigenetic modulation, nucleic acid detection, disease modeling, and more importantly for therapeutic purposes. CRISPR-based clinical and experimental methods have applications across a wide range of areas, especially for cancer research and, possibly, anticancer therapy. On the other hand, given the influential role of microRNAs (miRNAs) in the regulations of cellular division, carcinogenicity, tumorigenesis, migration/invasion, and angiogenesis in diverse normal and pathogenic cellular processes, in different stages of cancer, miRNAs are either oncogenes or tumor suppressors, according to what type of cancer they are involved in. Hence, these noncoding RNA molecules are conceivable biomarkers for diagnosis and therapeutic targets. Moreover, they are suggested to be adequate predictors for cancer prediction. Conclusive evidence proves that CRISPR/Cas system can be applied to target small non-coding RNAs. However, the majority of studies have highlighted the application of the CRISPR/Cas system for targeting protein-coding regions. In this review, we specifically discuss diverse applications of CRISPR-based tools for probing miRNA gene function and miRNA-based therapeutic involvement in different types of cancers.

Keywords: CRISPR/Cas; CRISPR–Cas9 library screen; Cancer therapy; Diagnostic; miRNA.

Fig. 1

The method of CRISPR/Cas9-mediated miRNA editing is shown in a schematic diagram. TheCas9 nuclease from S. pyogenesis was directed to genomic DNA, such as thehsa-miR-17 gene, via a sgRNA made up of a scaffold and a 20-nt guide sequence (red) (magenta). The DNA target (red line on top strand) and the guide sequence pair upstream of the necessary 50-NGG neighboring motif (PAM; green). Around 3 kb upstream of the PAM, the Cas9 nuclease causes a double-strand break (DSB) in the targeted genomic DNA (such as the Drosha or Dicer-processing sites) (red triangles). DNA repair mechanisms mediated by the host are used to fix the DSB caused by Cas9 nucleotide; bp base pairs. B CRISPR/cas9 can change the miRNA biogenesis process. The production of mature miRNAs in cells can be triggered by SgRNAs that target the sequences within or nearby Drosha- and Dicer-processing sites, respectively, in the secondary stem–loop structure of primary miRNA sequences. Red is used to represent both of the mature miR-17 sequence’s arms in the hairpin's stem–loop. PAM sequences are highlighted in green. The production of mature miRNAs in cells can be triggered by SgRNAs that target the sequences within or nearby Drosha- and Dicer-processing sites, respectively, in the secondary stem–loop structure of primary miRNA sequences. Red is used to represent both of the mature miR-17 sequence's arms in the hairpin's stem–loop. PAM sequences are highlighted in green (Aquino-Jarquin 2017)