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

Molecular therapies and functional studies greatly benefit from spatial and temporal precision of genetic intervention. We therefore conceived and explored tag-activated microRNA (miRNA)-mediated endogene deactivation (TAMED) as a research tool and potential lineage-specific therapy. For proof of principle, we aimed to deactivate gamma-globin repressor BCL11A in erythroid cells by tagging the 3 & PRIME; untranslated region (UTR) of BCL11A with miRNA recognition sites (MRSs) for the abundant erythromiR miR-451a. To this end, we employed nucleofection of CRISPR/Cas9 ribonucleoprotein (RNP) particles alongside double- or single-stranded oligodeoxynucleotides for, respectively, non-homologous-end-joining (NHEJ)- or homology-directed-repair (HDR)-mediated MRS insertion. NHEJ-based tagging was imprecise and inefficient (& LE;6%) and uniformly produced knock-in- and indel-containing MRS tags, whereas HDR-based tagging was more efficient (& LE;18%), but toxic for longer donors encoding concatenated and thus potentially more efficient MRS tags. Isolation of clones for robust HEK293T cells tagged with a homozygous quadruple MRS resulted in 25% spontaneous reduction in BCL11A and up to 36% reduction after transfection with an miR-451a mimic. Isolation of clones for human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells tagged with single or double MRS allowed detection of albeit weak gamma-globin induction. Our study demonstrates suitability of TAMED for physiologically relevant modulation of gene expression and its unsuitability for therapeutic application in its current form.

Automated summary

Spatial and temporal precision of genetic intervention greatly benefits molecular therapies and functional studies. This study explores tag-activated microRNA (miRNA)-mediated endogene deactivation for research and potential lineage-specific therapy. The results demonstrate the suitability of this method for physiologically relevant modulation of gene expression, but its current form is not suitable for therapeutic applications.