Jing Liu

In vivo genome correction holds promise for generating durable disease cures; yet, effective stem cell editing remains challenging. In this work, we demonstrate that optimized lung-targeting lipid nanoparticles (LNPs) enable high levels of genome editing in stem cells, yielding durable responses. Intravenously administered gene-editing LNPs in activatable tdTomato mice achieved >70% lung stem cell editing, sustaining tdTomato expression in >80% of lung epithelial cells for 660 days. Addressing cystic fibrosis (CF), NG-ABE8e messenger RNA (mRNA)-sgR553X LNPs mediated >95% cystic fibrosis transmembrane conductance regulator (CFTR) DNA correction, restored CFTR function in primary patient-derived bronchial epithelial cells equivalent to Trikafta for F508del, corrected intestinal organoids and corrected R553X nonsense mutations in 50% of lung stem cells in CF mice. These findings introduce LNP-enabled tissue stem cell editing for disease-modifying genome correction.

BACKGROUND: Due to the importance of Penicillium chrysogenum holding in medicine, the genome of low-penicillin producing laboratorial strain Wisconsin54-1255 had been sequenced and fully annotated. Through classical mutagenesis of Wisconsin54-1255, product titers and productivities of penicillin have dramatically increased, but what underlying genome structural variations is still little known. Therefore, genome sequencing of a high-penicillin producing industrial strain is very meaningful. RESULTS: To reveal more insights into the genome structural variations of high-penicillin producing strain, we sequenced an industrial strain P. chrysogenum NCPC10086. By whole genome comparative analysis, we observed a large number of mutations, insertions and deletions, and structural variations. There are 69 new genes that not exist in the genome sequence of Wisconsin54-1255 and some of them are involved in energy metabolism, nitrogen metabolism and glutathione metabolism. Most importantly, we discovered a 53.7 Kb "new shift fragment" in a seven copies of determinative penicillin biosynthesis cluster in NCPC10086 and the arrangement type of amplified region is unique. Moreover, we presented two large-scale translocations in NCPC10086, containing genes involved energy, nitrogen metabolism and peroxysome pathway. At last, we found some non-synonymous mutations in the genes participating in homogentisate pathway or working as regulators of penicillin biosynthesis. CONCLUSIONS: We provided the first high-quality genome sequence of industrial high-penicillin strain of P. chrysogenum and carried out a comparative genome analysis with a low-producing experimental strain. The genomic variations we discovered are related with energy metabolism, nitrogen metabolism and so on. These findings demonstrate the potential information for insights into the high-penicillin yielding mechanism and metabolic engineering in the future.