SA Genome Editing Facility

Caspase-2, one of the most evolutionarily conserved of the caspase family, has been implicated in maintenance of chromosomal stability and tumour suppression. Caspase-2 deficient ( Casp2 −/− ) mice develop normally but show premature ageing-related traits and when challenged by certain stressors, succumb to enhanced tumour development and aneuploidy. To test how caspase-2 protects against chromosomal instability, we utilized an ex vivo system for aneuploidy where primary splenocytes from Casp2 −/− mice were exposed to anti-mitotic drugs and followed up by live cell imaging. Our data show that caspase-2 is required for deleting mitotically aberrant cells. Acute silencing of caspase-2 in cultured human cells recapitulated these results. We further generated Casp2 C320S mutant mice to demonstrate that caspase-2 catalytic activity is essential for its function in limiting aneuploidy. Our results provide direct evidence that the apoptotic activity of caspase-2 is necessary for deleting cells with mitotic aberrations to limit aneuploidy.

Central conducting lymphatic anomaly (CCLA), characterized by the dysfunction of core collecting lymphatic vessels including the thoracic duct and cisterna chyli, and presenting as chylothorax, pleural effusions, chylous ascites, and lymphedema, is a severe disorder often resulting in fetal or perinatal demise. Although pathogenic variants in RAS/mitogen activated protein kinase (MAPK) signaling pathway components have been documented in some patients with CCLA, the genetic etiology of the disorder remains uncharacterized in most cases. Here, we identified biallelic pathogenic variants in MDFIC , encoding the MyoD family inhibitor domain containing protein, in seven individuals with CCLA from six independent families. Clinical manifestations of affected fetuses and children included nonimmune hydrops fetalis (NIHF), pleural and pericardial effusions, and lymphedema. Generation of a mouse model of human MDFIC truncation variants revealed that homozygous mutant mice died perinatally exhibiting chylothorax. The lymphatic vasculature of homozygous Mdfic mutant mice was profoundly mispatterned and exhibited major defects in lymphatic vessel valve development. Mechanistically, we determined that MDFIC controls collective cell migration, an important early event during the formation of lymphatic vessel valves, by regulating integrin β 1 activation and the interaction between lymphatic endothelial cells and their surrounding extracellular matrix. Our work identifies MDFIC variants underlying human lymphatic disease and reveals a crucial, previously unrecognized role for MDFIC in the lymphatic vasculature. Ultimately, understanding the genetic and mechanistic basis of CCLA will facilitate the development and implementation of new therapeutic approaches to effectively treat this complex disease.

Synaptic dysfunction is a manifestation of several neurobehavioral and neurological disorders. A major therapeutic challenge lies in uncovering the upstream regulatory factors controlling synaptic processes. Plant homeodomain (PHD) finger proteins are epigenetic readers whose dysfunctions are implicated in neurological disorders. However, the molecular mechanisms linking PHD protein deficits to disease remain unclear. Here, we generated a PHD finger protein 21B-depleted (Phf21b-depleted) mutant CRISPR mouse model (hereafter called Phf21bΔ4/Δ4) to examine Phf21b's roles in the brain. Phf21bΔ4/Δ4 animals exhibited impaired social memory. In addition, reduced expression of synaptic proteins and impaired long-term potentiation were observed in the Phf21bΔ4/Δ4 hippocampi. Transcriptome profiling revealed differential expression of genes involved in synaptic plasticity processes. Furthermore, we characterized a potentially novel interaction of PHF21B with histone H3 trimethylated lysine 36 (H3K36me3), a histone modification associated with transcriptional activation, and the transcriptional factor CREB. These results establish PHF21B as an important upstream regulator of synaptic plasticity-related genes and a candidate therapeutic target for neurobehavioral dysfunction in mice, with potential applications in human neurological and psychiatric disorders.

Abstract The RNA-guided endonuclease CRISPR-Cas system from Streptococcus pyogenes (SpCas9) is widely used for generating genetically modified mice via zygotic microinjection. Although SpCas9 is a potent mutagen, it requires an NGG proto-spacer adjacent motif (PAM) at the target site, restricting sequence targetability. Here, we show that RNA-guided endonucleases that utilize a range of alternative PAM sequences can edit the mouse genome at the neurog3 ( Ngn3 ) locus: SpCas9 VQR (NGAN PAM), SpCas9 VRER (NGCG), AsCas12a (TTTN), SaCas9 (NNGRRT), and SaCas9 KKH (NNNRRT). Additional experiments targeting tyrosinase and frizzled3 with SaCas9 KKH and its parent protein demonstrated that these endonucleases generated mutations in up to 100% of embryos across three loci. Remarkably, in contrast to wild-type SpCas9, these endonucleases frequently generated mutant embryos that retain unmodified alleles in both template-free and HDR-repair experiments. Our findings broaden PAM recognition options for mouse genome editing and identify SaCas9/SaCas9 KKH as useful alternatives when targeting genes with null lethal phenotypes.

The advent of CRISPR systems has transformed genome editing, offering unparalleled efficiency and versatility with wide therapeutic potential. However, conventional CRISPR systems face key limitations, including unpredictable and imprecise outcomes during repair of double-stranded breaks and reliance on specific protospacer adjacent motif sequences. In response, prime editing (PE) has emerged as a powerful alternative, enabling precise custom edits using a fusion of Cas9 nickase and an engineered reverse transcriptase (RT) together with a PE guide RNA (pegRNA) that encodes the desired repair template. PE enables edits to be installed at or downstream of the target site, expanding the range of targetable sequences. Since its inception, PE has undergone extensive optimization, including Cas variant selection, RT engineering, and pegRNA improvements. In parallel, advances in delivery, including nanoparticles and split viral systems, have accelerated translation across preclinical disease models. Notably, PE has now entered the clinic, with the first-in-human study reporting functional restoration with a promising safety profile to date. Here, we summarize recent mechanistic insights, architectural innovations, and therapeutic applications of PE and discuss the remaining challenges in efficiency, delivery, and safety that will shape broader clinical impact.

Enhancers are vitally important during embryonic development to control the spatial and temporal expression of genes. Recently, large scale genome projects have identified a vast number of putative developmental regulatory elements. However, the proportion of these that have been functionally assessed is relatively low. While enhancers have traditionally been studied using reporter assays, this approach does not characterise their contribution to endogenous gene expression. We have studied the murine Nestin (Nes) intron 2 enhancer, which is widely used to direct exogenous gene expression within neural progenitor cells in cultured cells and in vivo. We generated CRISPR deletions of the enhancer region in mice and assessed their impact on Nes expression during embryonic development. Loss of the Nes neural enhancer significantly reduced Nes expression in the developing CNS by as much as 82%. By assessing NES protein localization, we also show that this enhancer region contains repressor element(s) that inhibit Nes expression within the vasculature. Previous reports have stated that Nes is an essential gene, and its loss causes embryonic lethality. We also generated 2 independent Nes null lines and show that both develop without any obvious phenotypic effects. Finally, through crossing of null and enhancer deletion mice we provide evidence of trans-chromosomal interaction of the Nes enhancer and promoter.

Cardiac outflow tract morphogenesis requires coordinated interactions between multiple cell populations and is dependent on the contribution of cardiac progenitors from the second heart field. While neural crest cells have been proposed to impact second heart field development, how they regulate progenitor behaviour remains unclear. Here, we discover neural crest cells are a primary source of Dickkopf-1 (DKK1) which modulates Wnt signalling activity in the second heart field to influence the balance between cardiac progenitor maintenance and differentiation. We show that the ubiquitin ligase NEDD4 regulates DKK1, with disruption of Nedd4 leading to outflow tract defects. We further identify a new NEDD4 variant underlying human congenital heart disease. Our findings uncover an unexpected role for neural crest cells as a rheostat of Wnt signalling in cardiac progenitors, identifying a new molecular pathway promoting outflow tract morphogenesis, and a new causative factor of congenital heart disease.

Abstract Enhancers are vitally important during embryonic development to control the spatial and temporal expression of genes. Recently, large scale genome projects have identified a vast number of putative developmental regulatory elements. However, the proportion of these that have been functionally assessed is relatively low. While enhancers have traditionally been studied using reporter assays, this approach does not characterise their contribution to endogenous gene expression. We have studied the murine Nestin ( Nes) intron 2 enhancer, which is widely used to direct exogenous gene expression within neural progenitor cells in cultured cells and in vivo . We generated CRISPR deletions of the enhancer region in mice and assessed their impact on Nes expression during embryonic development. Loss of the Nes neural enhancer significantly reduced Nes expression in the developing CNS by as much as 82%. By assessing NES protein localization, we also show that this enhancer region contains repressor element(s) that inhibit Nes expression within the vasculature. Previous reports have stated that Nes is an essential gene, and its loss causes embryonic lethality. We also generated 2 independent Nes null lines and show that both develop without any obvious phenotypic effects. Finally, through crossing of null and enhancer deletion mice we provide evidence of trans -chromosomal interaction of the Nes enhancer and promoter.