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Megalith : studies in stone
\"Following the success of Quadrivium, Sciencia, Designa, and Trivium in the acclaimed Wooden Books series, Megalith is a compendium of writings about stone structures throughout history. How do you predict eclipses at Stonehenge? Why were stone monuments built where they are? What is the meaning of the designs in ancient rock art? In this lavishly illustrated volume, eight expert authors guide readers through the history of rock structures from Stonehenge to the stone circles in France, Poland, America, and Africa. These monuments appear around the globe, connecting the modern world and ancient times. Packed with detailed information and rare and exquisite engravings, woodcuts, and drawings, Megalith is a timeless and valuable sourcebook for our world's oldest buildings and our earliest visual art.\"--provided by publisher.
Book
Predicting the mutations generated by repair of Cas9-induced double-strand breaks
The DNA mutation produced by cellular repair of a CRISPR-Cas9-generated double-strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, but depend on DNA sequence at the targeted location. Here we systematically study the influence of flanking DNA sequence on repair outcome by measuring the edits generated by >40,000 guide RNAs (gRNAs) in synthetic constructs. We performed the experiments in a range of genetic backgrounds and using alternative CRISPR-Cas9 reagents. In total, we gathered data for >109 mutational outcomes. The majority of reproducible mutations are insertions of a single base, short deletions or longer microhomology-mediated deletions. Each gRNA has an individual cell-line-dependent bias toward particular outcomes. We uncover sequence determinants of the mutations produced and use these to derive a predictor of Cas9 editing outcomes. Improved understanding of sequence repair will allow better design of gene editing experiments.
Journal Article
CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice
Here, we show CRISPR/Cas9-based targeted somatic multiplexmutagenesis and its application for high-throughput analysis of gene function in mice. Using hepatic single guide RNA (sgRNA) delivery, we targeted large gene sets to induce hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). We observed Darwinian selection of target genes, which suppress tumorigenesis in the respective cellular/tissue context, such asPtenorCdkn2a,and conversely found low frequency ofBrca1/2alterations, explaining mutational spectra in human ICC/HCC. Our studies show that multiplexed CRISPR/Cas9 can be used for recessive genetic screening or high-throughput cancer gene validation in mice. The analysis of CRISPR/Cas9-induced tumors provided support for a major role of chromatin modifiers in hepatobiliary tumorigenesis, including that of ARID family proteins, which have recently been reported to be mutated in ICC/HCC. We have also comprehensively characterized the frequency and size of chromosomal alterations induced by combinatorial sgRNA delivery and describe related limitations of CRISPR/Cas9 multiplexing, as well as opportunities for chromosome engineering in the context of hepatobiliary tumorigenesis. Our study describes novel approaches to model and study cancer in a high-throughput multiplexed format that will facilitate the functional annotation of cancer genomes.
Journal Article
Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice
Mouse transgenesis has provided fundamental insights into pancreatic cancer, but is limited by the long duration of allele/model generation. Here we show transfection-based multiplexed delivery of CRISPR/Cas9 to the pancreas of adult mice, allowing simultaneous editing of multiple gene sets in individual cells. We use the method to induce pancreatic cancer and exploit CRISPR/Cas9 mutational signatures for phylogenetic tracking of metastatic disease. Our results demonstrate that CRISPR/Cas9-multiplexing enables key applications, such as combinatorial gene-network analysis,
in vivo
synthetic lethality screening and chromosome engineering. Negative-selection screening in the pancreas using multiplexed-CRISPR/Cas9 confirms the vulnerability of pancreatic cells to
Brca2
-inactivation in a
Kras
-mutant context. We also demonstrate modelling of chromosomal deletions and targeted somatic engineering of inter-chromosomal translocations, offering multifaceted opportunities to study complex structural variation, a hallmark of pancreatic cancer. The low-frequency mosaic pattern of transfection-based CRISPR/Cas9 delivery faithfully recapitulates the stochastic nature of human tumorigenesis, supporting wide applicability for biological/preclinical research.
CRISPR/Cas9 technology has been used for genome engineering
in vivo
. Here, the authors use a transfection technique to deliver multiple guide RNAs to the pancreas of adult mice, allowing genetic screening and chromosome engineering in pancreatic cancer.
Journal Article
PiggyBac mutagenesis and exome sequencing identify genetic driver landscapes and potential therapeutic targets of EGFR-mutant gliomas
Background
Glioma is the most common intrinsic brain tumor and also occurs in the spinal cord. Activating
EGFR
mutations are common in
IDH1
wild-type gliomas. However, the cooperative partners of
EGFR
driving gliomagenesis remain poorly understood.
Results
We explore
EGFR
-mutant glioma evolution in conditional mutant mice by whole-exome sequencing, transposon mutagenesis forward genetic screening, and transcriptomics. We show mutant
EGFR
is sufficient to initiate gliomagenesis in vivo, both in the brain and spinal cord. We identify significantly recurrent somatic alterations in these gliomas including mutant
EGFR
amplifications and
Sub1
,
Trp53
, and
Tead2
loss-of-function mutations. Comprehensive functional characterization of 96 gliomas by genome-wide
piggyBac
insertional mutagenesis in vivo identifies 281 known and novel
EGFR
-cooperating driver genes, including
Cdkn2a
,
Nf1
,
Spred1
, and
Nav3
. Transcriptomics confirms transposon-mediated effects on expression of these genes. We validate the clinical relevance of new putative tumor suppressors by showing these are frequently altered in patients’ gliomas, with prognostic implications. We discover shared and distinct driver mutations in brain and spinal gliomas and confirm in vivo differential tumor suppressive effects of
Pten
between these tumors. Functional validation with CRISPR-Cas9-induced mutations in novel genes
Tead2
,
Spred1
, and
Nav3
demonstrates heightened
EGFRvIII
-glioma cell proliferation. Chemogenomic analysis of mutated glioma genes reveals potential drug targets, with several investigational drugs showing efficacy in vitro.
Conclusion
Our work elucidates functional driver landscapes of
EGFR
-mutant gliomas, uncovering potential therapeutic strategies, and provides new tools for functional interrogation of gliomagenesis.
Journal Article
Correction to: PiggyBac mutagenesis and exome sequencing identify genetic driver landscapes and potential therapeutic targets of EGFR-mutant gliomas
Conditional PiggyBac transposase targeted to Rosa26 (tissue-specific PiggyBac transposase, TSPB), SA = splice acceptor; SD = splice donor; CAG = CAG promoter; SB = Sleeping Beauty; PB = PiggyBac inverted repeats; iPBase = insect version of the PiggyBac transposase. Gene inactivation can occur if the transposon inserts in the body of the gene as a consequence of gene trapping which can occur in either orientation because of the presence of two splice acceptors and bidirectional poly(A) (pA) sites. B. Outline of the experimental design: quadruple transgenic mice conditionally activate EGFRvIII expression and PiggyBac transposition in the central nervous system.
Journal Article
Compiler-Directed Regulation Enforcement for Unmanned Aerial Vehicles
As unmanned aerial vehicles (UAVs), often called “drones”, become more common in society, so do regulations and restrictions on their operation. Many of these regulations directly involve the conduct of the operator or operators of the UAVs, but some only involve the attitude, position, and trajectory of the UAV. This paper proposes that the latter type of regulations can and should be enforced by the framework governing the operation of the UAV itself to lift responsibility from the programmer or pilot. For autonomous aircrafts, this can be implemented as part of the language and enforced by the compiler. To this end, the open-source UAV framework Paparazzi has been extended to include compiler-driven enforcement of two distinct restrictions imposed by the Federal Aviation Administration (FAA), which regulates all air traffic in the United States, and lays groundwork for more. Our new features support the enforcement of the altitude limit and the ability to specify no-fly zones. We evaluate the effectiveness of our compiler-driven approach through Paparazzi simulation.
Dissertation
PiggyBac Transposon Mutagenesis: A Tool for Cancer Gene Discovery in Mice
Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here, we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon-transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed that PiggyBac is capable of genome-wide mutagenesis. The PiggyBac screen uncovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 21 mouse lines to be compatible with both transposon systems in constitutive, tissue- or temporal-specific mutagenesis. Mice with different transposon types, copy numbers, and chromosomal locations support wide applicability.
Journal Article
Genome-wide transposon screening and quantitative insertion site sequencing for cancer gene discovery in mice
Friedrich
et al.
describe their toolkit for transposon-based insertional mutagenesis in mice for discovering cancer genes. Genome-wide transposon insertion sites are identified, mapped and quantified using QiSeq.
Transposon-mediated forward genetics screening in mice has emerged as a powerful tool for cancer gene discovery. It pinpoints cancer drivers that are difficult to find with other approaches, thus complementing the sequencing-based census of human cancer genes. We describe here a large series of mouse lines for insertional mutagenesis that are compatible with two transposon systems,
PiggyBac
and
Sleeping Beauty
, and give guidance on the use of different engineered transposon variants for constitutive or tissue-specific cancer gene discovery screening. We also describe a method for semiquantitative transposon insertion site sequencing (QiSeq). The QiSeq library preparation protocol exploits acoustic DNA fragmentation to reduce bias inherent to widely used restriction–digestion-based approaches for ligation-mediated insertion site amplification. Extensive multiplexing in combination with next-generation sequencing allows affordable ultra-deep transposon insertion site recovery in high-throughput formats within 1 week. Finally, we describe principles of data analysis and interpretation for obtaining insights into cancer gene function and genetic tumor evolution.
Journal Article
A conditional piggyBac transposition system for genetic screening in mice identifies oncogenic networks in pancreatic cancer
Roland Rad and colleagues report development of a new conditional
piggyBac
transposition system for performing insertional mutagenesis screens in mice. They apply the system to identify new oncogenic driver pathways for pancreatic cancer.
Here we describe a conditional
piggyBac
transposition system in mice and report the discovery of large sets of new cancer genes through a pancreatic insertional mutagenesis screen. We identify Foxp1 as an oncogenic transcription factor that drives pancreatic cancer invasion and spread in a mouse model and correlates with lymph node metastasis in human patients with pancreatic cancer. The propensity of
piggyBac
for open chromatin also enabled genome-wide screening for cancer-relevant noncoding DNA, which pinpointed a
Cdkn2a cis
-regulatory region. Histologically, we observed different tumor subentities and discovered associated genetic events, including
Fign
insertions in hepatoid pancreatic cancer. Our studies demonstrate the power of genetic screening to discover cancer drivers that are difficult to identify by other approaches to cancer genome analysis, such as downstream targets of commonly mutated human cancer genes. These
piggyBac
resources are universally applicable in any tissue context and provide unique experimental access to the genetic complexity of cancer.
Journal Article