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

International Journal of Molecular Sciences

Yukio Nakamura¹  Panayiota L. Papasavva²  Marina Kleanthous²  Constantinos C. Loucari²  Carsten W. Lederer²  Petros Patsali²  Ryo Kurita³ 

[1] Cell Engineering Division, RIKEN BioResource Research Center, Tsukuba 305-0074, Japan;Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia 2371, Cyprus;Research and Development Department, Central Blood Institute, Blood Service Headquarters, Japanese Red Cross Society, Koto-ku, Tokyo 135-8521, Japan;

关键词: gene tagging;    cell/tissue-type-specific gene therapy;    γ-globin induction;    hemoglobinopathies;    BCL11A;    erythroid-specific;   

DOI  :  10.3390/ijms23031082

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown