Daheron, L. | bioRxiv Science

bioRxiv2025

Gene editing in "cell villages" enables exploring disease-relevant mutations in many genetic backgrounds

Understanding how individual genetic backgrounds shape the effects of disease-associated mutations is central to elucidating the biology of complex psychiatric disorders. We developed a scalable village editing strategy that enables simultaneous genome editing across multiple induced pluripotent stem cell (iPSC) lines, allowing systematic assessment of how polygenic context modulates the impact of specific mutations. Using pooled CRISPR editing in 15 iPSC lines spanning a range of schizophrenia (SCZ) polygenic risk scores, we generated homozygous and heterozygous knockouts in two known SCZ-associated genes: LRP1, involved in cholesterol import, and NRXN1, a presynaptic adhesion molecule. By mixing all lines prior to editing and de-multiplexing them afterward, we efficiently produced multi-donor knockout neurons at scale. Transcriptomic profiling revealed that LRP1 and NRXN1 loss produce both shared and donor-specific effects on neuronal gene expression, with variable perturbation of neurotransmitter transport and cholesterol biosynthesis pathways across genetic backgrounds. These results demonstrate that village editing enables systematic dissection of gene-background interactions in human neurons, offering a powerful framework for studying the polygenic architecture of psychiatric disease.

cell biology↗

bioRxiv2023

Monitoring hPSC genomic stability in the chromosome 20q region by ddPCR

Copy number increases involving chromosome 20q with gain of the gene BCL2L1 are a prevalent form of genomic instability in hPSC. In addition to large aneuploidies, findings in this region often include microamplifications that are too small to detect by G-banded karyotyping. Gene editing procedures warrant especially close monitoring of 20q genomic stability because they involve p53-activating stressors that select for the survival of BCL2L1-aneuploid cells. Here we describe an optimized strategy for detecting BCL2L1 copy number increases in hPSC cultures using duplexed droplet digital PCR (ddPCR) with genomic DNA or cell lysate as the starting material. The procedure consists of droplet generation, thermocycling, droplet reading and data analysis. The expected result is a copy number estimate derived by comparing the number of droplets positive for BCL2L1 to the number positive for a reference template, PVRL2. This procedure generates same-day screening results for 1 to 96 samples, providing a convenient option for screening hPSC cultures that is easily integrated into a gene editing workflow.

cell biology↗

bioRxiv2020

Extra-hematopoietic immunomodulatory role of the SCID-susceptibility gene DOCK-2 identified by stepwise maturation of human iPSCs into clonogenic mesodermal stromal progenitors

Stromal cells contribute to organ integrity as fibroblasts and to vascular stability as pericytes, in addition to their enigmatic niche function in many tissues. Their inherent immunomodulatory capacity attracted particular attention, initiating numerous clinical trials, particularly testing trophic regeneration and immunomodulation. Key stromal immune functions are still enigmatic. Here we show that dedicator of cytokinesis (DOCK-2) previously described for causing immune cell dysfunction plays a role in extra-hematopoietic immunity by regulating stromal/fibroblast immunomodulatory function. We used three independent strategies including iPSC-derived mesodermal stromal cell (MSC) lineage maturation, severe combined immunodeficiency (SCID) patient-derived cells and CRISPR/Cas9 knockout to support our findings. Human induced pluripotent stem cells (iPSCs) were generated from healthy bone marrow and umbilical cord blood-derived fibroblasts by Sendai virus-mediated transient expression of Yamanaka factors. After mesoderm induction, stromal differentiation was induced by platelet-derived growth factors under animal serum-free conditions. Under feeder-free defined conditions, iPSCs differentiated into expandable and cryo-preservable CD73+/CD105+/Tra-1-81- early iPS-MSCs lacking immunosuppressive potential. Successive maturation was required for reaching the canonical MSC phenotype and immunomodulatory competence over time, while maintaining clonogenicity, comparable to parental MSCs. Sequential RNAseq revealed acquisition of a spectrum of immune-related genes significantly expressed in mature iPS-MSCs and resembling parental MSCs immune gene expression. The DOCK-2 gene attracted our attention because mutations can cause SCID. Interestingly, SCID patient-derived fibroblast lines harboring bi-allelic DOCK-2 mutations showed significantly reduced immunomodulatory capacity compared to non-mutated control fibroblasts. CRISPR/Cas9-mediated DOCK-2 knockout in healthy iPSCs resulted in iPS-MSCs that also displayed reduced immunomodulatory capacity, thus confirming a role of DOCK-2 in stromal immune function. At a mechanistic level, DOCK-2 deficiency resulted in disturbed subcellular localization of CDC42. This provides first evidence for an extra-hematopoietic immunomodulatory role of DOCK-2 in stromal cells, previously considered restricted to hampering immune cell migration/function resulting in SCID. We may speculate that some of the signs and symptoms of persisting immune disease after successful hematopoietic stem cell transplantation in SCID patients could at least in part be due to mutations, like DOCK-2-/-, permissive outside the hematopoietic immune system, as evidenced also by the increased virus infection susceptibility of DOCK-2 deficient fibroblasts.

immunology↗