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.

Keywords: BCL11A; CD34+ cells; HUDEP-2 cells; cell/tissue-type-specific gene therapy; erythroid-specific; gene tagging; hemoglobinopathies; γ-globin induction.

Figure 1

Summary of detected erythromiRs in CD34+ and HUDEP-2 cells. (a) DNA Nanoball Small RNA Sequencing and DEGseq differential expression analysis identified 162 and 183 significantly upregulated miRNAs during erythroid differentiation in CD34+ and HUDEP-2 cells, respectively. A Venn diagram of these miRNAs revealed a set of 73 miRNAs common to both cell sources, likely representing key adult-type erythromiRs. (b) The top 20 erythromiRs were sorted by late-erythroid expression in CD34+ and HUDEP-2 cells (see Supplementary Table S1 for all 73 miRNAs). ¹: mean normalized miRNA counts in late-erythroid samples and ²: mean log₂ fold change of miRNA expression in late- vs. early-erythroid samples, both as calculated by DEGseq [37].

Figure 2

TAMED tools and modes of delivery. Potential integration sites (CRISPR/Cas9-mediated DSB sites) of MRSs are indicated in 5′ UTR (blue) and 3′ UTR (orange) of the BCL11A-XL isoform. Designs and delivery modes of blunt-ended dsODNs (bearing two or four tandem repeats of MRSs for miR-451a) and ssODNs (bearing one or two tandem repeats of MRSs for miR-451a) are schematically illustrated. Representative of all seven UTR sites, potential insertion events are only indicated for 3′ UTR 1, 593 nt upstream from the end of the BCL11A-XL long 3′ UTR (3206 nt). Shown with grey overlay are inverted integration events that will occur as undesirable side products for NHEJ-mediated tagging with blunt-ended dsODNs. DSB: double-strand break, dsODN: double-stranded oligodeoxynucleotide, HDR: homology-directed repair, MRS: miRNA recognition site, NHEJ: non-homologous end joining, P: phosphate group, ssODN: single-stranded oligodeoxynucleotide, UTR: untranslated region (light blue: 5′ UTR, orange: 3′ UTR), green boxes: non-UTR BCL11A-XL sequence elements, brown boxes: flanking genomic DNA elements.

Figure 3

Analysis of genome editing of 5′ and 3′ UTR of BCL11A-XL. (a) Representative gel images showing full-length PCR products and T7EI cleavage products (red brackets) of 5′ and 3′ UTR-edited sites. A Cas9-only transduced sample (EMPTY) was also analyzed in parallel to assess specificity of T7EI cleavage. (b) Quantification of T7EI assay was based on mean gray value of gel bands in ImageJ, using the 1–(1–(fraction cleaved))^1/2 formula [51] for 3′ UTR 1 (n = 3), 3′ UTR 2 (n = 1), 3′ UTR 3 (n = 1), 3′ UTR 4 (n = 2), 5′ UTR 1 (n = 3), 5′ UTR 2 (n = 2) and 5′ UTR 3 (n = 2). Error bars show the standard deviation of the sample mean. (c) Immunoblot analysis of BCL11A expression in samples edited with shortlisted gRNAs (red boxes in (b)) confirmed the neutral effect of editing on gene expression for at least two gRNAs, 3′ UTR 1 and 5′ UTR 1 (d) Chromatograms showing the absence of γ-globin in shortlisted samples (red boxes in (c)) except for SC, which showed 5.14-fold induction of γ-globin relative to EMPTY (peaks corresponding to γ-globin are indicated as ^Gγ and ^Aγ). EMPTY: Cas9-only-transduced sample, MOCK: mock-transduced cell sample, SC: cell sample transduced with gRNA targeting the start codon of BCL11A.

Figure 4

NHEJ-mediated integration of MRSs in HEK293T cells. (a) Gel image showing T7EI cleavage products (red brackets) of edited BCL11A-XL 3′ UTR 1 site. (b) Quantification of T7EI gel bands showing similar percentages of genome editing across samples (32.6% to 44.8%). (c) Apparent absence of PCR-RFLP HaeIII cleavage for test samples, indicative of dsODN integration being below the detection threshold. (d) Top: schematic illustration of dsODN-specific amplification assay at the 3′ UTR 1 site; bottom: amplicons of ~250 and ~302 bp revealing the presence of 2 MRSs and 4 MRSs in cells, respectively. Of note, undesirable inverted integration of MRSs into 3′ UTR 1 (see greyed-out part of Figure 2) is not detected by this assay. MOCK: mock-nucleofected cell sample, NO DONOR: cell sample nucleofected only with RNPs, MRS: miRNA recognition site, dsODN451-2MRSs/*: cell samples nucleofected with RNPs and dsODN bearing two miR451a MRSs at the indicated picomole quantity, dsODN451-4MRSs/5: cell sample nucleofected with RNPs and 5 pmole dsODN bearing four miR451a MRSs, +: a 994-bp PCR product giving cleavage products of 572 and 422 bp after digestion with HaeIII.

Figure 5

NHEJ-mediated integration of MRSs in HUDEP-2 and CD34+ cells. (a) Gel image showing T7EI cleavage products (red brackets) of edited BCL11A-XL 3′ UTR 1 site in HUDEP-2 cells. (b) Quantification of T7EI gel bands from (a), showing dsODN-concentration-correlated reduction in genome editing across samples. (c) Quantification of indel formation (genome editing efficiency) by TIDE for comparison with T7E1 assay results from (a). While showing slightly higher total efficiency for NO DONOR (77.4%) and lower efficiencies for all other samples (6–43.4%) than the T7E1 assay, TIDE gave the same overall trend of diminished total editing efficiency with increasing amount of donor. TIDE additionally demonstrated that the selected gRNA 3′ UTR 1 induced mainly deletions, with a 2-nt deletion as the most frequent event in the absence (28.4%) or presence (<2–13.7%) of donor DNA. (d) Dose-dependent dsODN-mediated cytotoxicity in HUDEP-2 cells, as measured by trypan blue assay 72 h after nucleofection. (e) Indicative of dsODN integration, a dsODN-specific amplification assay at the 3′ UTR 1 site gave ~250 bp products in both HUDEP-2 (left gel) and primary CD34+ cells (right gel). Separate gels are indicated by a dashed line. MOCK: mock-nucleofected cell sample, NO DONOR: cell sample nucleofected only with RNPs, MRS: miRNA recognition site, dsODN451-2MRSs/*: cell samples nucleofected with RNPs and dsODN bearing two miR451a MRSs at the indicated picomole quantity.

Figure 6

HDR-mediated integration of MRSs in HUDEP-2 cells. PCR-RFLP analysis of cells nucleofected with RNPs and ss DNA oligos was performed for the assessment of integration efficiency. Bands of 436 bp represent unmodified target sites, larger bands insertions with incomplete digestion by DdeI/HaeIII, and smaller bands cleavage products of insertions. All bands corresponding to insertions are indicated by arrowheads (band sizes of 554 bp and 277 bp for donors with 2 MRSs, and of 528 bp and 264 bp for donors with 1 MRS). Corresponding rates of DdeI/HaeIII cleavage in PCR-RFLP (%) (after subtraction of background average cleavage rates of control samples) are reported below the gels. (a) Analysis of cells nucleofected with RNPs and Ultramer DNA Oligos. (b) Analysis of cells nucleofected with Alt-R RNPs and Alt-R HDR-2MRSs DNA Oligo (left gel), shown in comparison with analysis of cells nucleofected with standard Cas9 RNPs and Ultramer ssODN451TS_2MRSs DNA Oligo (right gel). Separate gels are indicated by dashed lines. MOCK: mock-nucleofected sample, NO DONOR: cell sample nucleofected only with RNPs, MRS: miRNA recognition site, NTS: non-target strand, TS: target strand, +: PCR products bearing restriction sites for DdeI/HaeIII used as positive controls. See Section 4.2 and Supplementary Table S2 for ss DNA oligo naming.

Figure 7

Analysis of monoclonal cell populations at the DNA level. (a) Exemplary gel showing initial screening for positive HEK293T clones (clones with MRSs in the correct orientation), based on dsODN-specific amplification. Different sizes of PCR products correspond to integrations of different numbers of MRSs. (b) Sequencing chromatogram of the 3′ UTR 1 region for wild-type CONTROL HUDEP-2 clone. Representative of other sequencing data for 3′ UTR 1 clones, sequence trace data become mixed after successive long mononucleotide (T) runs. (c) Examples of imprecise insertion events for HEK293T clone D and HUDEP-2 clone A, representative of imprecisions for all detected NHEJ-based dsODN insertion events in both HEK293T and HUDEP-2 cells. For homozygous HEK293T clone D, alignment of its sequence trace with the predicted precise insertion of donor DNA revealed a 3-bp chromosomal deletion and insertion of at least three intact MRSs. For compound heterozygote HUDEP-2 clone A, corresponding alignments revealed a 9-bp chromosomal deletion in one allele and a 56-bp chromosomal deletion and a 48-bp insertion of intact MRSs in the other allele. Inadvertent deletions are indicated by red boxes.