Mutational History of a Human Cell Lineage from Somatic to Induced Pluripotent Stem Cells.

The accuracy of replicating the genetic code is fundamental. DNA repair mechanisms protect the fidelity of the genome ensuring a low error rate between generations. This sustains the similarity of individuals whilst providing a repertoire of variants for evolution. The mutation rate in the human genome has recently been measured to be 50-70 de novo single nucleotide variants (SNVs) between generations. During development mutations accumulate in somatic cells so that an organism is a mosaic. However, variation within a tissue and between tissues has not been analysed. By reprogramming somatic cells into induced pluripotent stem cells (iPSCs), their genomes and the associated mutational history are captured. By sequencing the genomes of polyclonal and monoclonal somatic cells and derived iPSCs we have determined the mutation rates and show how the patterns change from a somatic lineage in vivo through to iPSCs. Somatic cells have a mutation rate of 14 SNVs per cell per generation while iPSCs exhibited a ten-fold lower rate. Analyses of mutational signatures suggested that deamination of methylated cytosine may be the major mutagenic source in vivo, whilst oxidative DNA damage becomes dominant in vitro. Our results provide insights for better understanding of mutational processes and lineage relationships between human somatic cells. Furthermore it provides a foundation for interpretation of elevated mutation rates and patterns in cancer

Genome-wide CRISPR-KO Screen Uncovers mTORC1-Mediated Gsk3 Regulation in Naive Pluripotency Maintenance and Dissolution

Summary: The genetic basis of naive pluripotency maintenance and loss is a central question in embryonic stem cell biology. Here, we deploy CRISPR-knockout-based screens in mouse embryonic stem cells to interrogate this question through a genome-wide, non-biased approach using the Rex1GFP reporter as a phenotypic readout. This highly sensitive and efficient method identified genes in diverse biological processes and pathways. We uncovered a key role for negative regulators of mTORC1 in maintenance and exit from naive pluripotency and provided an integrated account of how mTORC1 activity influences naive pluripotency through Gsk3. Our study therefore reinforces Gsk3 as the central node and provides a comprehensive, data-rich resource that will improve our understanding of mechanisms regulating pluripotency and stimulate avenues for further mechanistic studies. : Li et al. conducted genome-wide CRISPR screens in mouse ESCs to identify genes affecting maintenance of and exit from naive pluripotency using a Rex1GFP reporter. They show that loss of two mTORC1-negative regulators, Tsc1/2 and Gator1, can cause opposing phenotypes through differential regulation of Gsk3 activity. Keywords: CRISPR, screening, naive pluripotency, exit from pluripotency, Akt, mTORC1, mTORC2, GATOR1, Nprl2, Tsc

A resource of vectors and ES cells for targeted deletion of microRNAs in mice.

The 21-23 nucleotide, single-stranded RNAs classified as microRNAs (miRNA) perform fundamental roles in diverse cellular and developmental processes. In contrast to the situation for protein-coding genes, no public resource of miRNA mouse mutant alleles exists. Here we describe a collection of 428 miRNA targeting vectors covering 476 of the miRNA genes annotated in the miRBase registry. Using these vectors, we generated a library of highly germline-transmissible C57BL/6N mouse embryonic stem (ES) cell clones harboring targeted deletions for 392 miRNA genes. For most of these targeted clones, chimerism and germline transmission can be scored through a coat color marker. The targeted alleles have been designed to be adaptable research tools that can be efficiently altered by recombinase-mediated cassette exchange to create reporter, conditional and other allelic variants. This miRNA knockout (mirKO) resource can be searched electronically and is available from ES cell repositories for distribution to the scientific community

Mutation rate of human pluripotent cells in culture.

<p><b>a</b>. The mean numbers of SNVs accumulated during 60 cell divisions in 2 iPSC lines, S7-RE14 (<i>n</i> = 3) and S4-SF6 (<i>n</i> = 2) and a human ESC line H9 (<i>n</i> = 3). Data are shown as mean ± SD. <b>b</b>. Mutation rate per cell per division in each pluripotent cell line.</p

Summary of WGS and deep sequence analysis.

<p>Summary of WGS and deep sequence analysis.</p

Mutational signatures <i>in vivo</i>, <i>in vitro</i> and through reprogramming.

<p><b>a</b>. Schematic showing the longitudinal progression from <i>in vivo</i> development, <i>in vitro</i> culture of somatic cells through reprogramming and finally through to the experimental set-up used to calculate the mutation rate in iPSC maintenance culture. <b>b</b>. Mutational spectrum of SNVs found in the EPCs (top), and primary (middle) and sub-cloned (bottom) S7-RE14 iPSC lines. Clonal (left) and sub-clonal (right) mutations were shown separately. <b>c</b>. Contribution of mutational processes identified by the NNMF analysis. The germ line mutations described in ref 18 were analysed [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005932#pgen.1005932.ref018" target="_blank">18</a>]. The NNMF analysis was not performed for the mutations in sub-clonal S7-EPC due to the limited number of mutations available.</p

Comparing acquired SNVs in iPSCs derived from a polyclonal or a monoclonal origin.

<p><b>a</b>. Schematic comparing the reprogramming of polyclonal and monoclonal cells. Polyclonal cells such as fibroblasts (left panel) give rise to iPSCs which do not share a majority of mutations since they are derived from different progenitors. In contrast, iPSCs derived from monoclonal cells (right panel) such as EPCs share a proportion of their mutations and carry private mutations specific to each line. <b>b, c</b>. Exome sequencing of iPSCs generated using fibroblasts from two different individuals, a 65-year-old alpha-1 antitrypsin deficiency patient (AATD) (<b>b</b>) and a healthy subject, S2 (<b>c</b>). The data for iPSC-B were taken from our previous work [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005932#pgen.1005932.ref011" target="_blank">11</a>]. Each column represents one SNV in the indicated gene. Duplicated genes indicate two adjacent SNVs. Green, mutation absent; pink, mutation present. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005932#pgen.1005932.s004" target="_blank">S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005932#pgen.1005932.s005" target="_blank">S3</a> Tables for the complete description. <b>d, e</b>. Exome sequencing of iPSC lines generated using monoclonal EPCs from the same AATD patient in <b>b</b> as well as a healthy subject, S7 (<b>e</b>). Orange, mutation detected by amplicon resequencing. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005932#pgen.1005932.s006" target="_blank">S4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005932#pgen.1005932.s007" target="_blank">S5</a> Tables for the complete description.</p