Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system

What is it about?

Facing more and more human novel genetic variants revealed by high-throughput DNA sequencing, it is highly desirable to develop a suitable animal model to verify these variants at a systems level for better sequence interpretation and variant classification, and thus for better genomic medicine. The true diploid frog Xenopus tropicalis shows high efficiency and penetrance in modeling human cancers as revealed by recent apc and rb knockout studies. While the majority of human genetic diseases arise from point mutations, the established homology-independent knock-in method in frogs is suboptimal for modeling those diseases. Recent studies revealed that CRISPR/Cas9-coupled cytidine deaminase is effective in single nucleotide editing in cell lines and various model animals and plants. It remains to be investigated if this hybrid works in Xenopus tropicalis. Here we have optimized the experimental conditions and for the first time demonstrate that the CRISPR/Cas9D10A-AID/APOBEC-UGI base editor is an efficient and robust tool for precise C to T conversion in Xenopus tropicalis. The editing efficiency is so high that germ line transmission was readily achieved without any pre-selection of founders. Recapitulation of human Holt-Oram syndrome and oculocutaneous albinism type 1A via targeting the conserved tbx5 R237 and tyr C28 sites, respectively, demonstrates the beauty of this diploid frog and this strategy in modeling human illness caused by single base alterations. Together, our study makes it possible to rapidly, efficiently, and cost-effectively model many of the C to T or G to A human genetic variants in frogs at a systems level.

Featured Image