CRISPR Editing Enables Consequential Tag-Activated MicroRNA-Mediated Endogene Deactivation.
Panayiota L PapasavvaPetros PatsaliConstantinos C LoucariRyo KuritaYukio NakamuraMarina KleanthousCarsten Werner LedererPublished in: International journal of molecular sciences (2022)
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 γ-globin repressor BCL11A in erythroid cells by tagging the 3' 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 (≤6%) and uniformly produced knock-in- and indel-containing MRS tags, whereas HDR-based tagging was more efficient (≤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 γ-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.
Keyphrases
- crispr cas
- genome editing
- induced apoptosis
- cord blood
- gene expression
- cell proliferation
- long non coding rna
- cell cycle arrest
- randomized controlled trial
- genome wide
- long noncoding rna
- dna repair
- helicobacter pylori
- dna methylation
- endoplasmic reticulum stress
- dna damage
- signaling pathway
- oxidative stress
- label free
- real time pcr