Jensen, U. B. | bioRxiv Science

bioRxiv2018

Circularization of genes and chromosome by CRISPR in human cells

Extrachromosomal circular DNA (eccDNA) and ring chromosomes are genetic alterations found in humans with genetic disorders and diseases such as cancer. However, there is a lack of genetic engineering tool to recapitulate these features. Here, we report the discovery that delivery of pairs of CRISPR/Cas9 guide RNAs into human cells generate functional eccDNAs and ring chromosomes. We generated a dual-fluorescence eccDNA biosensor system, which allows us to study CRISPR deletion, inversion, and circularization of genes inside cells. Analysis after CRISPR editing at intergenic and genic loci in human embryonic kidney 293T cells and human mammary fibroblasts reveal that CRISPR deleted DNA readily form eccDNA in human cells. DNA in sizes from a few hundred base pairs up to a 47.4 megabase-sized ring chromosome (chr18) can be circularized. Our discoveries advance and expand CRISPR-Cas9 technology applications for genetic engineering, modeling of human diseases, and chromosome engineering.\n\nOne Sentence Summary: CRISPR circularization of DNA offers new tools for studying eccDNA biogenesis, function, chromosome engineering, and synthetic biology.

genomics↗

bioRxiv2018

2-APB arrests human keratinocyte proliferation and inhibits cutaneous squamous cell carcinoma in vitro

BackgroundThe epidermis is a stratified epithelium whose differentiation program is triggered in part by calcium. Dysregulation of keratinocyte differentiation may lead to non-melanoma skin cancers, including cutaneous squamous cell carcinoma (cSCC). The compound 2-aminoethoxydiphenyl borate (2-APB) modulates calcium signaling by altering activity of calcium-permeable channels of the transient receptor potential (TRP) and ORAI families, and is therefore poised to govern signaling pathways that control the balance of keratinocyte proliferation and differentiation.\n\nObjectiveWe sought to determine whether 2-APB alters differentiation of normal human keratinocytes and progression of human cSCCs models in vitro.\n\nMethodsPrimary human keratinocyte cultures were treated with 2-APB and levels of proliferation (EdU incorporation) and differentiation markers [quantitative PCR (qPCR)] were assessed. Human cSCC biopsies and cell lines were analyzed for TRP and ORAI gene expression via qPCR. cSCC cell lines were cultured in organtypic cultures and analyzed for growth and invasiveness after 2-APB or vehicle treatment.\n\nResultsCulturing human keratinocytes with 2-APB arrested cell proliferation, triggered differentiation-gene expression and altered epidermal stratification, indicating that 2-APB application is sufficient to promote differentiation. In human organotypic cSCC cultures, 2-APB attenuated tumor growth and invasiveness. Finally, expression of a panel of 2-APB-targeted ion channels (TRPV3, TRPV1, TRPC1, OraI1, OraI2 and OraI3) was dysregulated in high-risk cSCC biopsies.\n\nConclusionsCollectively, these findings identify 2-APB as a potential therapeutic for high-risk cSCCs.

cell biology↗