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New Report Demonstrates CRISPR for microRNA Inhibition

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A team of Chinese researchers this month reported a technique for blocking specific microRNAs based on CRISPR, a genome-editing technique derived from an immunity mechanism discovered in bacteria.

According to the study's authors, their approach can be easily adapted to target different miRNAs, can block the expression of miRNA clusters, and does not trigger off-target effects.

In bacteria, CRISPR — short for clustered regularly interspaced short palindromic repeats — works when a short DNA sequence known as a spacer is derived from a virus and incorporated into the bacterial genome, where it acts as a sort of memory of the infection.

Reinfection triggers the creation of CRISPR RNA (crRNA), single-stranded RNA that is complementary to the spacer sequence. Processed into its mature form by trans-activating crRNA (tracrRNA), crRNA acts as a guide for a complex containing the nuclease Cas9, which cuts both strands of the spacer's target DNA.

Recently, the CRISPR pathway as been adapted as a tool for selective gene engineering, wherein a specially designed crRNAs/tracrRNAs duplex dubbed guide RNA (gRNA) can direct Cas9 to a DNA sequence of interest, generating the double-strand breaks (DSBs) that cause targeted gene silencing. Notably, this method has proven effective in eukaryotic cells, including human cells.

Now, investigators from Northeast Forest University (NFU) and Jilin University have adapted this method for silencing miRNAs, according to a paper that appeared in Scientific Reports this week.

While miRNAs can be inhibited with complementary antisense oligonucleotides, such molecules typically require "expensive modifications" and usually generate off-target effects, the study's authors wrote. "In addition, the generation of loss-of-function miRNA mutations via homologous recombination is technically difficult because the transcripts are usually short."

Looking for an alternative to antisense, the research group turned to CRISPR, noting that DSBs in the loop region of a pre-miRNA should theoretically affect miRNA maturation during Drosha and Dicer processing.

To test their idea, they generated three exogenous shRNAs to serve as substitutes for miRNAs. The scientists then created three 3T3 reporter cell lines that expressed a doxycycline-inducible shRNA and a Venus-sensor fusion protein containing the target shRNA region.

According to the paper, crRNAs were designed to separately recognize the linker between the loop and seed region of the different shRNAs in the three vectors. After transfection of the vectors separately into the corresponding reporter cell lines, the team observed a "significant enhancement" of fluorescence, suggesting that inhibition of the targeted shRNAs results from specific DSBs in the target regions.

To test the CRISPR method against endogenous miRNAs, the researchers selected two monocistronic miRNAs: miR-21, which is highly expressed in 3T3 cells, and miR-30a, which contains the same looped backbone as the previously tested shRNAs, they wrote in Scientific Reports.

"After transfection with the corresponding repressive vector, both miRNAs were efficiently silenced," showing that miRNA expression can be suppressed in mouse cells by cutting the genome at a single site with Cas9.

A paper from another group published last year described a modified CRISPR interference system (CRISPRi) that could selectively and reversibly control gene expression on a genome-wide scale in Escherichia coli, and which could be modified for mammalian cells.

With an eye toward testing the reversibility of their CRISPR-based miRNA inhibition approach, the Jilin and NFU researchers analyzed the fluorescence intensity at different time points in one of the shRNA reporter cell lines from their first experiments and found that the signal reached a maximum at 72 hours, then decreased to baseline after another 48 hours, according to their study. No off-target effects were observed in the cells.

Aiming to test their method against miRNA clusters, the investigators targeted the miR-17-92 cluster — which includes miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92-1 — in mouse bone marrow stem cells using a crRNA designed to localize to the upstream region of miR-17. Focusing on miR-19a, miR-20a, and miR-92-1 to determine knockdown efficiency, the team found that all three miRNAs were repressed 48 hours after transfection with their crRNA.

Lastly, the scientists demonstrated that their approach is effective in porcine cells. However, they were unable to replicate the dose-dependent silencing effects observed by other groups using CRISPR for gene knockdown, instead finding that increasing the dose of Cas9/gRNA led to "severe toxicity."

It is unclear what caused the toxicity, but they added that their method will require further refinement for use in porcine cells in the future.

Overall, the findings indicate that CRISPR can be adapted to target a specific miRNA sequence by "simply switching the crRNA in a single repression vector," which only involves changing a 20 base-pair sequence, the study's authors wrote.

"Compared with other strategies, the CRISPR/CRISPRi system is easier to engineer and exhibits improved flexibility as a tool to analyze miRNA function and for future disease therapy," they concluded.

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