Spontaneous Single-Copy Loss of TP53 in Human Embryonic Stem Cells Markedly Increases Cell Proliferation and Survival

15Human pluripotent stem cells (hPSCs) generate a wide variety of disease-relevant cells that can 16 be used to improve the translation of preclinical research. Despite the potential of hPSCs, their 17 use for genetic screening has been limited because of technical challenges. We developed a 18 renewable Cas9/sgRNA-hPSC library where loss-of-function mutations can be induced at will. 19Our inducible-mutant hPSC library can be used for an unlimited number of genome-wide 20 screens. We screened for novel genes involved in 3 of the fundamental properties of hPSCs: 21Their ability to self-renew/survive, their capacity to differentiate into somatic cells, and their 22 inability to survive as single-cell clones. We identified a plethora of novel genes with unidentified 23 roles in hPSCs. These results are available as a resource for the community to increase the 24 understanding of both human development and genetics. In the future, our stem cell library 25 approach will be a powerful tool to identify disease-modifying genes. 27 VISUAL ABSTRACT 28All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Mammalian sirtuin 6 (Sirt6) is a conserved NAD+-dependent deacylase and mono-ADP ribosylase that is known to be involved in DNA damage repair, metabolic homeostasis, inflammation, tumorigenesis, and aging. Loss of Sirt6 in mice results in accelerated aging and premature death within a month. Here, we show that haploinsufficiency (i.e., heterozygous deletion) of Trp53 dramatically extends the lifespan of both female and male Sirt6-deficient mice. Haploinsufficiency of Trp53 in Sirt6-deficient mice rescues several age-related phenotypes of Sirt6-deficient mice, including reduced body size and weight, lordokyphosis, colitis, premature senescence, apoptosis, and bone marrow stem cell decline. Mechanistically, SIRT6 deacetylates p53 at lysine 381 to negatively regulate the stability and activity of p53. These findings establish that elevated p53 activity contributes significantly to accelerated aging in Sirt6-deficient mice. Our study demonstrates that p53 is a substrate of SIRT6, and highlights the importance of SIRT6-p53 axis in the regulation of aging.

Human pluripotent stem cells (hPSCs) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with acquisition of large copy number variants (CNVs) that provide mutant cells with a growth advantage in culture1–3. However, the nature, extent, and functional impact of other acquired genome sequence mutations in cultured hPSCs is not known. Here, we sequenced the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hESC) lines, including 26 lines prepared for potential clinical use4. We then applied computational strategies for identifying mutations present in a subset of cells5. Though such mosaic mutations were generally rare, we identified five unrelated hESC lines that carried six mutations in the TP53 gene that encodes the tumor suppressor P53. Notably, the TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We used droplet digital PCR to demonstrate that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that P53 mutation confers selective advantage. When we then mined published RNA sequencing data from 117 hPSC lines, we observed another nine TP53 mutations, all resulting in coding changes in the DNA binding domain of P53. Strikingly, in three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from loss of heterozygosity at the TP53 locus. As the acquisition and favored expansion of cancer-associated mutations in hPSCs may go unnoticed during most applications, we suggest that careful genetic characterization of hPSCs and their differentiated derivatives should be carried out prior to clinical use.