CRISPR Editing Enables Consequential Tag-Activated MicroRNA-Mediated Endogene Deactivation.

Area of Science:

• Gene regulation
• Molecular biology
• Biotechnology

Background:

• Precise spatial and temporal control of gene expression is crucial for molecular therapies and functional studies.
• MicroRNA (miRNA)-mediated gene regulation offers a powerful mechanism for targeted gene silencing.

Purpose of the Study:

• To develop and evaluate tag-activated miRNA-mediated endogene deactivation (TAMED) as a novel research tool and potential lineage-specific therapy.
• To assess the feasibility of deactivating the γ-globin repressor BCL11A in erythroid cells using TAMED.

Main Methods:

• CRISPR/Cas9 ribonucleoprotein (RNP) nucleofection was used to insert miRNA recognition sites (MRSs) into the 3' UTR of BCL11A.
• Both non-homologous end-joining (NHEJ) and homology-directed repair (HDR) pathways were employed for MRS insertion.
• HEK293T and human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells were utilized for proof-of-principle experiments.

Main Results:

• NHEJ-based tagging was inefficient (≤6%) and imprecise, yielding indel-containing MRS tags.
• HDR-based tagging was more efficient (≤18%) but exhibited toxicity with longer donor sequences.
• TAMED demonstrated a spontaneous reduction of BCL11A in HEK293T cells (25%) and up to 36% reduction with miR-451a mimic transfection.
• Weak γ-globin induction was observed in HUDEP-2 cells following TAMED, indicating limited gene modulation.

Conclusions:

• TAMED is a viable tool for physiologically relevant gene expression modulation in research settings.
• The current iteration of TAMED is unsuitable for direct therapeutic applications due to efficiency and toxicity concerns.