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

Abstract

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.

High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis

“…Base editing technology was exploited to target γ-globin modifiers as potential therapy for β-hemoglobinopathies. Use of HUDEP-2 cells, with their minimal baseline γ-globin expression, facilitates such analyses ( Papasavva et al, 2022 ), but owing to low transfection efficiency and high toxicity of plasmids in these sensitive cells, calls for in vitro transcribed mRNA in combination with commercial gRNA for delivery of BEs.…”

In search of an ideal template for therapeutic genome editing: A review of current developments for structure optimization

“…However, reducing the dsODN's length does not seem to increase insertion frequency (Wen et al, 2021) and does not enhance efficiency of genome editing. Moreover, addition of phosphorothioate linkages at the ends of the DNA strands helps to stabilize ODNs in cells, ensure nuclease resistance and facilitate ligation reactions, and both 5′ and 3′ end-phosphorylated dsODNs show higher integration frequencies in comparison with the dsODNs with 5′ end protection alone (Tsai et al, 2015;Papasavva et al, 2022).…”

Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi

“… 60 With shared interest in hemoglobinopathies, mutation-specific repair and fetal hemoglobin induction, Carsten W. Lederer (The Cyprus Institute of Neurology & Genetics, Nicosia, Cyprus) also introduced the concept of tag-activated microRNA (miRNA)-mediated endogene deactivation (TAMED) as a potentially general therapy and research approach. 61 TAMED draws on targeted insertion of miRNA recognition site (MRS) tags and on endogenous miRNA expression to achieve lineage-specific silencing of tagged endogenes. For the abundant erythroid miR-451a and the use case of BCL11A downregulation based on tagging with miR-451a cognate MRSs, Lederer’s team established proof of principle for TAMED, while concluding that therapeutically relevant efficiencies and wider application will depend on improved donor chemistries.…”