CTNNB1-dependent molecular pathways are disrupted proportional to gene-dosage in a human embryonic stem cell model of early neural differentiation

CTNNB1 encodes β-catenin, a multifunctional protein which acts as a WNT responsive transcriptional co-activator in the nucleus, and separately as a component of cell-cell adhesion complexes. Heterozygous loss-of-function variants of CTNNB1 cause a defined neurodevelopmental disorder. To explore the gene-dosage dependent effects of CTNNB1 on embryonic neural development, we performed CRISPR/Cas9-mediated genome editing of the H1 human embryonic stem cell line to engineer either mono- or bi-allelic CTNNB1 null alleles. During directed neural differentiation, bi-allelic CTNNB1 knockout lines displayed an overt prematurity of neurogenesis which was subtly mirrored in the mono-allelic loss lines. RNA sequencing revealed impact on the expression of genes involved in both cell-cell adhesion, mediated through cadherins and protocadherins, as well as WNT signaling. These stem cells with loss of CTNNB1 provide human-specific insight into the early role of CTNNB1 in neural differentiation We aimed to model haploinsufficiency of CTNNB1 in hESCs using CRISPR/Cas9 genome editing in the most frequently studied hESC line, H1. We introduced bi-allelic 77 bp frameshifting deletions into exon 3 of CTNNB1 in hESC using two independent sgRNAs. To generate hESC clones that retain one wild-type copy of CTNNB1, we performed homology-directed repair to correct a mutant CTNNB1 allele (NM_001904.4: c.184delG:p.Val62Serfs*18) that was found in one of the sequenced compound heterozygous clones (initially presumed to be a mono-allelic CTNNB1 77 bp deletion), back to the wild-type allele. to investigate the impact of heterozygous and complete CTNNB1 KO on the early stages of neural development. Three wild-type empty vector controls, heterozygous and complete CTNNB1 KO clones each, along with the H1 parental hESC line, were directed to differentiate into cortical neurons for 26 days using dual SMAD inhibition. The experiments were performed in two batches with replication of one heterozygous CTNNB1 clone #20 and one complete CTNNB1 KO clone #9 between each batch. We performed RNA-sequencing on the parental H1 hESC line (n=1), empty vector control (n=3), heterozygous (n=3) and complete CTNNB1 KO (n=3) clones at the undifferentiated (ESC; day 0), neuroepithelial sheet (NE; day 10), neural rosette (NR; day 13) and neural progenitor cell (NPC; day 23) stages of neural differentiation.