Journal: medRxiv

Article Title: Ultra-rare biallelic THAP12 variants cause loss of function and underlie severe epileptic encephalopathy

doi: 10.64898/2026.02.27.26347078

Figure Lengend Snippet: (A) Sanger sequencing confirms compound heterozygous variants in THAP12 in both affected siblings, with a maternally inherited frameshift (c.312del, red arrow) and a paternally inherited missense variant (c.829C>A, black arrow). (B) The two variants affect conserved residues within protein domains, particularly a proline at position 277 in the DUF4371 domain, as shown in a multi-species alignment (red arrow; Hs: Homo sapiens; Mm: Mus musculus; Rn: Rattus norvegicus; Xt: Xenopus tropicalis; Dr: Danio rerio ). (C) A Sashimi plot of RNA-seq reads from patients’ primary fibroblasts across the THAP12 locus shows no major changes in exon usage or alternative splicing between probands and their parents. Read counts on the splice junction arcs indicate the number of split reads supporting each exon-exon connection in each sample. (D) Structural modelling with AlphaFold3 predicts that THAP12 forms homodimers primarily through interactions between DUF4371 domains (left). In a THAP12-DNA complex predicted using AlphaFold3, the N-terminal THAP zinc-finger domains interacts with DNA using an electropostive surface (right). (E) The maternally inherited frameshift variant (Glu105AsnfsTer2) is predicted to truncate the protein after residue 106, abolishing the DUF4371 domain and likely impairing dimerization. The N-terminal segment (residues 1-106) is shown in color, corresponding to the truncated product of the frameshift allele. The paternally inherited Pro277Thr missense variant affects a conserved residue buried within the hydrophobic core of the DUF4371 domain, likely disrupting local folding and protein stability. (F-G) THAP12 transcript levels are not significantly changed in patient fibroblasts compared to parental controls, as shown by RNA-seq and qPCR analyses. (H-I) In contrast, THAP12 protein levels in patient fibroblasts are significantly reduced in both probands, as shown by Western blot and quantification. (J) Volcano plot displaying differentially expressed genes from bulk RNA-sequencing analysis of patient-derived fibroblasts compared to parental controls. Downregulated genes include several involved in neuronal and synaptic function (e.g., TUBB, RIMS1, GABRA3, KCNMB1 ). Significance is color-coded according to the - log10(adjusted p-value). The full list of differentially expressed genes is provided in Table S1. (K) Pathway enrichment analysis of differentially expressed genes highlights over-representation of pathways such as “Neuronal System”, “Signal Transduction”, and “Transmission across Chemical Synapses”. Dot size indicates the number of genes in each pathway; color represents -log10(p-value). Statistical analyses in panels F, G, and I used unpaired two-tailed Student’s t-test: *p < 0 . 05, **p < 0 . 01, ***p < 0 . 001, ****p < 0 . 0001; ns, not significant .

Article Snippet: Whole genome sequencing and analysis and RNA sequencing data were provided the Broad Institute Center for Mendelian Genomics (CMG) and were funded by the National Human Genome Research Institute (NHGRI) grants UM1HG008900 (with additional support from the National Eye Institute, and the National Heart, Lung and Blood Institute), R01HG009141, U01HG011755, and in part by the Chan Zuckerberg Initiative Donor-Advised Fund at the Silicon Valley Community Foundation grants 2019-199278, 2020-224274 (https://doi.org/10.37921/236582yuakxy) (funder DOI 10.13039/100014989).

Techniques: Sequencing, Variant Assay, RNA Sequencing, Alternative Splicing, Residue, Western Blot, Derivative Assay, Transduction, Transmission Assay, Two Tailed Test

Journal: medRxiv

Article Title: Ultra-rare biallelic THAP12 variants cause loss of function and underlie severe epileptic encephalopathy

doi: 10.64898/2026.02.27.26347078

Figure Lengend Snippet: (A) Schematic comparison of the human variants identified in the two affected siblings (top) with the corresponding genetically engineered mouse alleles (bottom), showing equivalent positions across conserved domains. (B) Structural alignment of the mouse and human THAP12 proteins, whose structures were predicted using AlphaFold3, reveals high overall similarity and highlights the position of the conserved Pro273 residue (orthologous to human Pro277) within a similar hydrophobic core of the DUF4371 domain. (C) Sanger sequencing confirms the genotypes of the three engineered alleles in mice (red arrows): insT, Glu105Asnfs*2 and Pro273Thr . (D) Representative images of E12.5 mouse embryos show that homozygous Pro273Thr mutants are severely developmentally delayed compared to wild-type and heterozygous littermates. (E) Table summarizing genotype frequencies at different developmental stages in intercrosses between heterozygous carriers. The absence of homozygous animals at birth confirms early embryonic lethality. Of note, data from both insT and Glu105Asnfs*2 alleles are gathered as “KO” in this panel. Full data for each stage and genotype are provided in Table S2. (F) Western blot of E12.5 embryonic heads shows reduced THAP12 protein levels in Pro273Thr homozygous mutants. (G) Quantification of THAP12 signal relative to wild-type confirms a significant decrease in homozygous embryos. Data are expressed as fold change relative to wild-type levels. Statistical analyses in panel G used a o ne-way ANOVA with Tukey post-hoc test: *p < 0 . 05, **p < 0 . 01, ***p < 0 . 001 .

Article Snippet: Whole genome sequencing and analysis and RNA sequencing data were provided the Broad Institute Center for Mendelian Genomics (CMG) and were funded by the National Human Genome Research Institute (NHGRI) grants UM1HG008900 (with additional support from the National Eye Institute, and the National Heart, Lung and Blood Institute), R01HG009141, U01HG011755, and in part by the Chan Zuckerberg Initiative Donor-Advised Fund at the Silicon Valley Community Foundation grants 2019-199278, 2020-224274 (https://doi.org/10.37921/236582yuakxy) (funder DOI 10.13039/100014989).

Techniques: Comparison, Residue, Sequencing, Western Blot

Journal: medRxiv

Article Title: Ultra-rare biallelic THAP12 variants cause loss of function and underlie severe epileptic encephalopathy

doi: 10.64898/2026.02.27.26347078

Figure Lengend Snippet: (A-B) Dorsal views of Tg[ elavl3 :GFP] larvae at 2 dpf (A) and 5 dpf (B) showing a reduced brain size in thap12 CRISPant compared to sham-injected controls. The lack of a clear midbrain-hindbrain boundary (dotted line) is indicated by asterisks. (C-E) Transverse sections immunostained for elavl3 show reduced brain size in thap12 CRISPant embryos at 2 dpf ( C ) and at 5 dpf ( D, E ). (F) Dorsal view of 3 dpf brains immunostained for acetylated tubulin showing reduced density of axonal tracts in thap12 CRISPant , especially at the level of the commissure (asterisks). (G-H) Quantification of brain area from whole-brain imaging of Tg[elavl3:GFP] larvae at 2 dpf (G) and 5 dpf (H) confirms a significant reduction of brain size in thap12 CRISPant . (I-J) thap12 CRISPant larvae show reduced numbers of elavl3 + neurons from immunolablled cross-sections (I) and commissural axonal tracts (J). (K) Volcano plot displaying differentially expressed genes from bulk RNA-sequencing analysis of microdissected larval brains from 4dpf thap12a -/- compared to wild-type siblings. Upregulated genes include several involved in apoptosis (e.g. tp53 ) and cell cyle (e.g. ccng1 ). Significance is color-coded according to the -log10(adjusted p-value). (L) Pathway enrichment analysis identifies p53 signaling, cell cycle, and metabolic stress as significantly enriched pathways in mutants compared to wild-type siblings. Dot size indicates the number of genes in each pathway; color represents -log10(p-value). (M) Acridine orange staining reveals increased cell death in the brain of thap12 CRISPant at 1 dpf. (N) Anti-phospho-H3 immunostaining shows a reduced number of proliferating cells in thap12 CRISPRant larvae at 1 dpf. Quantification are shown in panel O and P. (Q-T) Injection of human wild-type THAP12 mRNA, but not Pro277Thr mutant mRNA, rescues reduced proliferation in thap12 CRISPant larvae at 1 dpf (Q-R) and partially rescues brain size at 2 dpf (S-T) . The lack of a clear midbrain-hindbrain boundary (dotted line) described in panel A is indicated by asterisks. Scale bars are shown on each panel. Statistical analyses in panel G-J and O-P used unpaired two-tailed Student’s t-test, and in panels R and T used one-way ANOVA: *p < 0 . 05, **p < 0 . 01, ***p < 0 . 001, ****p < 0 . 0001; ns, not significant. fb: forebrain; mb: midbrain; hb: hindbrain; mhb: midbrain-hindbrain boundary (dotted line); bs: brainstem; ey: eye; re: retina; tec: tectum; teg: tegmentum; hl: hypothalamus .

Article Snippet: Whole genome sequencing and analysis and RNA sequencing data were provided the Broad Institute Center for Mendelian Genomics (CMG) and were funded by the National Human Genome Research Institute (NHGRI) grants UM1HG008900 (with additional support from the National Eye Institute, and the National Heart, Lung and Blood Institute), R01HG009141, U01HG011755, and in part by the Chan Zuckerberg Initiative Donor-Advised Fund at the Silicon Valley Community Foundation grants 2019-199278, 2020-224274 (https://doi.org/10.37921/236582yuakxy) (funder DOI 10.13039/100014989).

Techniques: Injection, Imaging, RNA Sequencing, Staining, Immunostaining, Mutagenesis, Two Tailed Test

Journal: Molecular cell

Article Title: ZC3H14 facilitates backsplicing by binding to exon-intron boundary and 3' UTR.

doi: 10.1016/j.molcel.2024.10.001

Figure Lengend Snippet: Figure 1. Genome-wide CRISPR screen for regulators of circRNA biogenesis with circReporter cells (A) Illustration of circReporter cell construction. The circGFP minigene included circVN1R1 flanking sequences, inverted split-GFP fragments, and IRES. (B) GFP and mCherry protein levels in circReporter cells treated with siRNA (sicircGFP) against circGFP backsplicing junction (BSJ). siNC, negative control siRNA with scrambled sequences. (C) Procedure of genome-wide CRISPR knockout screen. (D) Overlapped positive and negative regulators identified from GFPhigh and GFPlow cells. (E) GO analysis of 83 overlapped regulators identified from GFPhigh and GFPlow cells. (F) 20 regulators enriched in the RNA-binding GO term.

Article Snippet: Genome-wide CRISPR knockout screen and data analysis The Human CRISPR knockout pooled library (Brunello) was purchased from the Addgene (https://www.addgene.org/).

Techniques: Genome Wide, CRISPR, Negative Control, Knock-Out, RNA Binding Assay

Journal: Genome Research

Article Title: Genome-scale identification of cellular pathways required for cell surface recognition

doi: 10.1101/gr.231183.117

Figure Lengend Snippet: A cell-based genome-scale CRISPR-KO approach identifies pathways required for monoclonal antibody surface epitope recognition. ( A ) Schematic of the approach based on CRISPR/Cas9 technology using a genome-scale lentiviral gRNA library. Genes identified as being required for surface display of the epitope recognized by an anti-CD58 ( B ) and anti-GYPA ( C ) mAbs. The enrichment of gRNAs targeting each gene is quantified as the robust rank aggregation (RRA) score calculated using the MAGeCK software between selected cells that had lost the mAb epitope versus control cells and is shown plotted in rank order. ( D ) Genes identified as being required for surface display of the epitope recognized by an anti-CD59 mAb; here, genes are ordered alphabetically for clarity. Circles represent individual genes and are sized according to their false-discovery rate (FDR): large circle = FDR < 1%, small circle = 1% < FDR < 5%. Genes encoding the direct receptors are indicated with gray circles. Only genes with FDR < 5% are named and are color-coded according to their function.

Article Snippet: The Human Improved Genome-Wide Knockout CRISPR Library v1 (Addgene no. 67989), lentiviral Cas9 reporter plasmids pKLV2-U6gRNA5(gGFP)-PGKBFP2AGFP-W (Addgene no. 67980) and pKLV2-U6gRNA5(Empty)-PGKBFP2AGFP-W (Addgene no. 67979), lentiviral vector expressing Cas9 fused with the blasticidin-resistant gene at the C terminus pKLV2-EF1a-Cas9Bsd-W (Addgene no. 68343), and lentiviral CRISPR gRNA expression vector pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W (Addgene no. 67974) were used in this study.

Techniques: CRISPR, Software, Control

Journal: bioRxiv

Article Title: CRISPR activation screens map the genomic landscape of cancer glycome remodeling

doi: 10.1101/2025.05.26.656133

Figure Lengend Snippet: A) Inhibition of anticancer immunity by Siglec receptors. Siglecs bind to sialic acid-containing glycans on the surface of target cells. B) Components of the Siglec-7 ligand structure. Disialyl-T glycans are organized in dense arrays on the backbone of specific mucin-type O-glycoproteins. C ) Targeted overexpression of genes by CRISPRa. A cell line expressing a dCas9-GCN4 peptide array and a VP64-SunTag fusion protein serves as the target cell line (K-562-CRISPRa cells). This cell line is lentivirally transduced with an sgRNA targeting the promoter region of a target gene. Subsequent recruitment of chromatin remodeling factors induces an increase in transcriptional activity. D) Recombinant Siglec-Fc proteins were precomplexed with an AlexaFluor647-antihuFc antibody (1 μg/mL) for 1 hour on ice. Precomplexes were subsequently incubated with K-562 cells for 30 minutes and analyzed by flow cytometry. Representative flow cytometry plots are depicted for each Siglec-Fc as well as a human Fc (hFc) negative control. E) K-562-CRISPRa cells were lentivirally transduced with a genome-wide library of ∼104,000 sgRNAs (5 sgRNAs/gene). After selection and propagation, 1.25 x 10 8 cells were then stained with Siglec-Fc reagents as in B . A high-binding (top 20%) and low-binding (bottom 20%) of the fluorescent population was then selected and sorted by FACS.

Article Snippet: The CRISPRa-v2 library (top 5 sgRNAs/gene) containing 104,535 sgRNAs was purchased from Addgene (Pooled Library 83978).

Techniques: Inhibition, Over Expression, Expressing, Peptide Microarray, Transduction, Activity Assay, Recombinant, Incubation, Flow Cytometry, Negative Control, Genome Wide, Selection, Staining, Binding Assay

Journal: bioRxiv

Article Title: CRISPR activation screens map the genomic landscape of cancer glycome remodeling

doi: 10.1101/2025.05.26.656133

Figure Lengend Snippet: A) K-562-dCas9-CRISPRa cells were transduced with two different sgRNAs targeting the CD24 promoter region. After selection with puromycin, cells were stained with Siglec-10-Fc as in 1B and with a fluorescent antibody against CD24. Representative flow cytometry plots for both stains are shown. B) THP-1 cells were stained with Siglec-10-Fc as in 1B and a fluorescent antibody against CD24. Representative flow cytometry plots for both stains are shown. C) OCI-AML-2-Cas9 cells were lentivirally transduced with an sgRNA targeting MGAT1 to generate a polyclonal cell population containing WT and MGAT1 KO cells. Cells were then co-stained with Siglec-10-Fc and the lectin L-PHA (5 μg/mL), which binds to complex N-linked glycans. Representative staining is indicated on the flow cytometry plot. D) Graph indicates MFI of Siglec-10-Fc staining in the WT (L-PHA+) and MGAT1 KO (L-PHA-) populations. E) OCI-AML-2-Cas9 cells were lentivirally transduced with an sgRNA targeting C1GALT1. Cells were co-stained with Siglec-10-Fc and the lectin DBA, which binds to exposed α-GalNAc. Representative staining is indicated on the flow cytometry plot. F) Graph indicates MFI of Siglec-10-Fc staining in the WT (DBA-) and C1GALT1 KO (DBA+) populations. G) Pathway diagram indicates the enzymes involved in 2,3-linked and 2,6-linked sialylation of N-linked glycans. ST3GAL4, ST3GAL6 and ST6GAL1 were all strong hits in the Siglec-10 screen. H) The heat map indicates the average mRNA expression of key glycosyltransferase hits in cell lines derived from B-ALL, T-ALL, AML and multiple myeloma. mRNA expression values were extracted from DepMap and the Human Protein Atlas. I) OCI-AML-2-Cas9 cells were transduced an sgRNAs against ST3GAL4. Cells were then stained with fluorescently labeled Siglec-10-Fc. A representative flow cytometry plot is shown. J) The MFI of Siglec-10 staining in OCI-AML-2 WT and ST3GAL4 KO cells is indicated. MFI is internally normalized to the WT cell line. K) MM1S-Cas9 cells were transduced an sgRNAs against ST6GAL1. ST6GAL1 KO cells were isolated by FACS using the lectin SNA. Cells were then stained with fluorescently labeled Siglec-10-Fc. A representative flow cytometry plot is shown. L) The MFI of Siglec-10 staining in MM1S WT and ST6GAL1 KO cells is indicated. MFI is internally normalized to the WT cell line. M) K-562-CRISPRa cells were transduced and stained as in 4C . A representative flow cytometry plot is shown comparing Siglec-10-Fc staining in cells transduced with a non-targeting (NT) sgRNA to cells within the “CD44 High” expression gate. N) The MFI of Siglec-10-Fc staining in NT cells vs. “CD44 High” cells is shown. MFI is internally normalized to the WT cell line. O) K-562 CRISPRa cells were transduced with an sgRNA against CSPG4 and co-stained with Sig10-Fc and a CSPG4 antibody as in 4C . A representative flow cytometry plot is shown comparing Siglec-10-Fc staining in cells transduced with a non-targeting (NT) sgRNA to cells within the “CSPG4 High” expression gate. P) The MFI of Siglec-10 staining in NT cells vs. “CSPG4 High” cells is shown. MFI is internally normalized to the WT cell line. Statistical significance was determined using a two-tailed t-test. ** indicates p<0.01, * indicates p<0.05. Mean values plotted, error bars indicate SEM.

Article Snippet: The CRISPRa-v2 library (top 5 sgRNAs/gene) containing 104,535 sgRNAs was purchased from Addgene (Pooled Library 83978).

Techniques: Transduction, Selection, Staining, Flow Cytometry, Expressing, Derivative Assay, Labeling, Isolation, Two Tailed Test