Targeting miRNA by CRISPR/Cas in cancer: advantages and challenges

Abstract

Clustered regulatory interspaced short palindromic repeats (CRISPR) has changed biomedical research and provided entirely new models to analyze every aspect of biomedical sciences during the last decade. In the study of cancer, the CRISPR/CRISPR-associated protein (Cas) system opens new avenues into issues that were once unknown in our knowledge of the noncoding genome, tumor heterogeneity, and precision medicines. CRISPR/Cas-based gene-editing technology now allows for the precise and permanent targeting of mutations and provides an opportunity to target small non-coding RNAs such as microRNAs (miRNAs). However, the development of effective and safe cancer gene editing therapy is highly dependent on proper design to be innocuous to normal cells and prevent introducing other abnormalities. This study aims to highlight the cutting-edge approaches in cancer-gene editing therapy based on the CRISPR/Cas technology to target miRNAs in cancer therapy. Furthermore, we highlight the potential challenges in CRISPR/Cas-mediated miRNA gene editing and offer advanced strategies to overcome them.

Keywords: CRISPR; CRISPR/Cas12; CRISPR/Cas9; Cancer therapy; Gene editing; miRNAs.

Fig. 1

A graphical illustration of miRNA biogenesis. The pre-miRNAs have one or more incomplete hairpin structures that have a stem of about 33 base pairs. Ribonucleases Drosha and Dicer process the pri-miRNA precursor in two separate processes. In the nucleus, Drosha first cuts the pri-miRNA into a pre-miRNA about 70 nucleotides in length, which is then transferred to the cytoplasm by XPO5. The mature, functional, ds miRNA is then processed by Dicer using the pre-miRNA as a template. After maturation, the miRNA is covalently linked to RISC, a multiprotein complex that contains the AGO protein and is essential for RNA silencing. Exon 1 and exon 2 are connected together when the RNA splicing process takes place and leads to the formation of the lariat RNA (circular molecules with a short tail). Following RNA splicing and additional processing, the intron-containing spliced lariat may function as a pri-miRNA for intronic miRNA synthesis. XPO5 exportin-5, ds double-stranded, RISC RNA-induced silencing complex, AGO argonaute, ADAR adenosine deaminase RNA specific, TRBP tar RNA-binding protein, EGFR epidermal growth factor receptor

Fig. 2

A graphical illustration of how oncogenic and tumor suppressor miRNAs are regulated during tumorigenic events. a When oncogenic miRNAs are expressed at higher levels in malignant cells, tumor suppressor gene expression is lowered either as a result of mRNA degradation or hypermethylation. b Oncogenic miRNA expression may be increased by decreasing the expression level of tumor suppressor miRNAs. Both oncogenic and tumor suppressor miRNAs contribute to tumorigenesis by promoting a variety of malignant phenotypes, including cell development, anti-apoptotic activity, invading, angiogenic, and spreading. RISC RNA-induced silencing complex, ORF open reading frame

Fig. 3

Therapeutic use of miRNAs in cancer treatment. miRNA replacement therapies or oncogenic miRNA inhibition are the two primary current methods utilized to prevent the overexpression and functions of miRNAs. miRNA replacement therapy such as ligand conjugated miRNAs, liposomes, miRNA mimics, and viral vectors are used to suppress the oncogenic miRNAs. Furthermore, miRNA inhibition therapy includes small molecule inhibitors, miRNA sponge, antisense, and CRISPR/Cas9 which inhibit the oncogenic miRNA’s function. miRNAs microRNAs, CRISPR/Cas9 clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9

Fig. 4

The CRISPR/Cas system and gene editing mechanism. a CRISPR/Cas locus in the bacterial genome with associated transcription and translation products. b CRISPR/Cas engineered for site-specific gene editing. c Editing of dsDNA using CRISPR/Cas12a and CRISPR/Cas9 respectively. CRISPR/Cas clustered regularly interspaced short palindromic repeats/CRISPR-associated protein, dsDNA double strands DNA, crRNA CRISPR RNA, TracrRNA trans-activating CRISPR RNA, sgRNA single-guide RNA, PAM protospacer adjacent motif

Fig. 5

An illustration shows how Cas9 stabilizes the R-loop. In order to construct an R-loop structure, the targeted strand of DNA must link with the 20-nt of gRNA as the DNA curves. The sgRNA not matched to a target DNA sequence (0-nt RNA: DNA) that has a complementary sequence to the gRNA. DNA binding at a sequence matching the 20-nt sgRNA helps proteins accept the RNA–DNA helix and displaced non-target DNA strand. Complete R-loop formation constitutes the signal for the subsequent structure of the targeted gene. gRNA guide RNA, nt nucleotide, sgRNA single-guide RNA, Cas9 CRISPR-associated protein 9, PAM protospacer adjacent motif, Nuc nuclease, Rec recognition

Fig. 6

An illustration showed different targeting DNA regions of the post-transcriptional editing of miRNAs with the CRISPR/Cas system. Targeting terminal loops: CRISPR/Cas9 has the potential to inhibit the production of monoisotopic miRNAs by targeting either the terminal loop or the 5ʹ region of the pre-miRNA. Targeting secondary stem loop: Random targeting of pri-miRNA at secondary stem-loop structure by CRISPR/Cas9. Targeting mature miRNA: CRISPR/Cas9 sequences that target mature miRNA are used to successfully inhibit miRNA expression. Dicer cleavage sites: The expression levels of mature miRNAs can be successfully down-regulated by gRNA when it has been directed to target miRNA in the Dicer site. Drosha cleavage sites: gRNA successfully targets mature miRNAs at the Drosha region to bring down the expression levels of certain miRNAs. CRISPR/Cas clustered regularly interspaced short palindromic repeats/CRISPR-associated protein, gRNA guide RNA, sgRNA single-guide RNA, crRNA CRISPR RNA

Fig. 7

An illustration shows applying Ca12a instead of Cas9 protein, which has the ability to recognize PAM sequences in miRNA editing. By altering the miR-21 coding sequences in glioma cells, CRISPR/Cas12a decreases miR-21 expression through microenvironment cells, which controls both in vitro and in vivo cell proliferation. CRISPR clustered regularly interspaced short palindromic repeats, PAM protospacer adjacent motif, sgRNA single-guide RNA

Fig. 8

An illustration shows the main strategy to overcome multiple sites targeting or one site targeting through the CRISPR/Cas system. The CRISPR/Cas system targets one miRNA, which in turn suppresses miRNA to bind numerous genes and limit the synthesis of tumor protein. The CRISPR/Cas system is designed to target numerous miRNAs, which in turn restrict the same oncogenic gene, thereby limiting the growth of tumors. The CRISPR/Cas system is intended to target a large number of miRNAs, which then block the activity of the same oncogenic gene. As a result, the progression of tumor cell is suppressed or slowed down. CRISPR clustered regularly interspaced short palindromic repeats, sgRNA single-guide RNA, Cas CRISPR-associated protein