Biology subjects

Cornean, A.

Publications and source records attributed to Cornean, A..

5 recordsLinked to original sources

bioRxiv2026

Cytosine base editing workflow for quality-controlled multiplex-knockout hiPSC lines

Dissecting polygenic disease mechanisms requires human cell models that harbour multiple targeted genetic modifications in a defined background. However, generating and rigorously validating such models remains difficult. We developed a cytosine base editing workflow to generate multiplex-knockout (KO) human induced pluripotent stem cell (hiPSC) lines. First, we assessed six cytosine base editor (CBE) variants and selected evoBE4max. We then combined sgRNA-guided introduction of premature termination codons and splice-site mutations with fluorescence-based enrichment. This yielded a median on-target C-to-T editing efficiency of 77.5% (range, 27.0-86.5%) across six loci. We generated single-, double-, and triple-KO hiPSC lines for endolysosomal Ca{superscript 2} signalling components (OCaR2, TPC1, TPC2) and confirmed loss-of-function at transcript and protein levels. We performed extensive quality control, including pluripotency assessment, SNP-array karyotyping, and whole-genome sequencing, which indicated minimal guide-directed off-target editing. We further extended multiplex editing to ORAI Ca{superscript 2} channel paralogs. This framework supports scalable production of quality-controlled multiplex-KO hiPSC lines.

genetics↗

bioRxiv2025

Precise generation of bystander-free mouse models with ABE9-SpRY.

Point mutations cause many genetic disorders, but modelling them in organisms is technically challenging. Creating mouse models that mimic these mutations is crucial for establishing a causal relationship between mutations and disease phenotype, thereby supporting the development of therapeutic strategies. Adenine base editors (ABEs) can correct single-nucleotide variants (SNVs) in disease modelling without double-stranded breaks (DSBs) or donor DNA, achieving higher product purity than traditional Cas9 methods. Earlier ABE techniques faced issues like limited targetability, bystander editing, and off-target effects. By combining two editor advancements, we introduced and tested ABE9-SpRY, an improved ABE variant fused with a PAM-flexible SpRY-Cas9 nickase. Our results show that ABE9-SpRY effectively generates three out of four targeted A-to-G mutations in mouse embryos, with significantly fewer off-target effects than ABE8e-SpRY, achieving desired editing efficiencies of up to 96% in individual adult founder mice.ABE9-SpRY also enhances product purity in mouse embryos and human induced pluripotent stem cells (hiPSCs) compared to ABE8e-SpRY. Our findings showcase ABE9-SpRYs precision and versatility, highlighting it as a powerful tool for accurate in vivo point mutation modelling.

genetics↗

bioRxiv2023

Streamlined generation of CRISPR/Cas9-mediated single-cell knockout clones in murine cell lines.

Clonal cell lines harbouring loss-of-function mutations in genes of interest are crucial for studying the cellular functions of the encoded proteins. Recent advances in genome engineering have converged on the CRISPR/Cas9 technology to quickly and reliably generate frameshift mutations in the target genes across various cell lines and species. Although high on-target cleavage efficiencies can be obtained reproducibly, screening and identifying clones with loss-of-function alleles remains a major bottleneck. Here, we describe a single sgRNA strategy to generate CRISPR/Cas9-mediated frameshift mutations in target genes of mammalian cell lines that can be easily and cost-effectively identified. Given the proliferation of workhorse cell lines such as HEK293 and N2a cells and the resulting clonal expansion of the cell type, our protocol can facilitate the isolation of knockout clonal cell lines and their genetic validation within a period of down to 3-4 weeks.

cell biology↗

bioRxiv2021

Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction

Single nucleotide variants (SNVs) are prevalent genetic factors shaping individual trait profiles and disease susceptibility. The recent development and optimizations of base editors, rubber and pencil genome editing tools now promise to enable direct functional assessment of SNVs in model organisms. However, the lack of bioinformatic tools aiding target prediction limits the application of base editing in vivo. Here, we provide a framework for adenine and cytosine base editing in medaka (Oryzias latipes) and zebrafish (Danio rerio), ideal for scalable validation studies. We developed an online base editing tool ACEofBASEs (a careful evaluation of base-edits), to facilitate decision-making by streamlining sgRNA design and performing off-target evaluation. We used state-of-the-art adenine (ABE) and cytosine base editors (CBE) in medaka and zebrafish to edit eye pigmentation genes and transgenic GFP function with high efficiencies. Base editing in the genes encoding troponin T and the potassium channel ERG faithfully recreated known cardiac phenotypes. We finally validated missense mutations in novel candidate genes of congenital heart disease (CHD) dapk3, ube2b, usp44, and ptpn11 and present the resulting heart specific phenotypes. This base editing framework applies to a wide range of SNV-susceptible traits accessible in fish, facilitating straight-forward candidate validation and prioritization for detailed mechanistic downstream studies.

genetics↗

bioRxiv2021

hei-tag: a highly efficient tag to boost targeted genome editing

Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems1. While active in all species tested so far, editing efficiencies still leave room for improvement. To reach its target, the bacterial Cas9 needs to be efficiently shuttled into the nucleus as attempted by fusion of nuclear localization signals (NLSs) to the Cas9 protein2. Additional domains such as FLAG- or myc-tags are added for immediate detection or straight-forward purification3. To avoid steric hinderance impacting on activity, amino acid linkers are employed connecting Cas9 and additional domains. We present the hei-tag (high efficiency-tag), boosting the activity of the wide variety of CRISPR/Cas genome editing tools. The addition of the hei-tag to Cas9 or a C-to-T base editor dramatically enhances the respective targeting efficiency in model systems ranging from fish to mammals, including tissue culture applications. This allows to instantly upgrade existing and potentially highly adapted systems as well as establish novel highly efficient tools.

genetics↗