There is, I think, something ethereal about i —the square root of minus one. I remember first hearing about it at school. It seemed an odd beast at that time—an intruder hovering on the edge of reality.

Usually familiarity dulls this sense of the bizarre, but in the case of i it was the reverse: over the years the sense of its surreal nature intensified. It seemed that it was impossible to write mathematics that described the real world in …

I and i

Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

/pdf/multiplex-genome-engineering-using-crispr-cas-systems-2qm4sjme5b.pdf

Multiplex Genome Engineering Using CRISPR/Cas Systems

BACKGROUND Most patients with non-small-cell lung cancer have no response to the tyrosine kinase inhibitor gefitinib, which targets the epidermal growth factor receptor (EGFR). However, about 10 percent of patients have a rapid and often dramatic clinical response. The molecular mechanisms underlying sensitivity to gefitinib are unknown. METHODS We searched for mutations in the EGFR gene in primary tumors from patients with non-small-cell lung cancer who had a response to gefitinib, those who did not have a response, and those who had not been exposed to gefitinib. The functional consequences of identified mutations were evaluated after the mutant proteins were expressed in cultured cells. RESULTS Somatic mutations were identified in the tyrosine kinase domain of the EGFR gene in eight of nine patients with gefitinib-responsive lung cancer, as compared with none of the seven patients with no response (P<0.001). Mutations were either small, in-frame deletions or amino acid substitutions clustered around the ATP-binding pocket of the tyrosine kinase domain. Similar mutations were detected in tumors from 2 of 25 patients with primary non-small-cell lung cancer who had not been exposed to gefitinib (8 percent). All mutations were heterozygous, and identical mutations were observed in multiple patients, suggesting an additive specific gain of function. In vitro, EGFR mutants demonstrated enhanced tyrosine kinase activity in response to epidermal growth factor and increased sensitivity to inhibition by gefitinib. CONCLUSIONS A subgroup of patients with non-small-cell lung cancer have specific mutations in the EGFR gene, which correlate with clinical responsiveness to the tyrosine kinase inhibitor gefitinib. These mutations lead to increased growth factor signaling and confer susceptibility to the inhibitor. Screening for such mutations in lung cancers may identify patients who will have a response to gefitinib.

/pdf/activating-mutations-in-the-epidermal-growth-factor-receptor-4a7r2t8eq0.pdf

Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib

Genome Editing Clustered regularly interspaced short palindromic repeats (CRISPR) function as part of an adaptive immune system in a range of prokaryotes: Invading phage and plasmid DNA is targeted for cleavage by complementary CRISPR RNAs (crRNAs) bound to a CRISPR-associated endonuclease (see the Perspective by van der Oost). Cong et al. (p. 819, published online 3 January) and Mali et al. (p. 823, published online 3 January) adapted this defense system to function as a genome editing tool in eukaryotic cells. A bacterial genome defense system is adapted to function as a genome-editing tool in mammalian cells. [Also see Perspective by van der Oost] Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

Chromatin, the physiological template of all eukaryotic genetic information, is subject to a diverse array of posttranslational modifications that largely impinge on histone amino termini, thereby regulating access to the underlying DNA. Distinct histone amino-terminal modifications can generate synergistic or antagonistic interaction affinities for chromatin-associated proteins, which in turn dictate dynamic transitions between transcriptionally active or transcriptionally silent chromatin states. The combinatorial nature of histone amino-terminal modifications thus reveals a “histone code” that considerably extends the information potential of the genetic code. We propose that this epigenetic marking system represents a fundamental regulatory mechanism that has an impact on most, if not all, chromatin-templated processes, with far-reaching consequences for cell fate decisions and both normal and pathological development.

/pdf/translating-the-histone-code-l291mddcr9.pdf

Translating the Histone Code

/pdf/trogocytosis-based-cell-platform-for-tcr-ligand-discovery-aljuadvc5s.pdf

Trogocytosis-based cell platform for TCR ligand discovery

Molecular Properties of Voltage-Gated Ion Channels

The determination of individual cell trajectories through a high-dimensional cell-state space is an outstanding challenge, with relevance towards understanding biological changes ranging from cellular differentiation to epigenetic (adaptive) responses of diseased cells to drugging. We report on a combined experimental and theoretic method for determining the trajectories that specific highly plastic BRAFV600E mutant patient-derived melanoma cancer cells take between drug-naive and drug-tolerant states. Recent studies have implicated non-genetic, fast-acting resistance mechanisms are activated in these cells following BRAF inhibition. While single-cell highly multiplex omics tools can yield snapshots of the cell state space landscape sampled at any given time point, individual cell trajectories must be inferred from a kinetic series of snapshots, and that inference can be confounded by stochastic cell state switching. Using a microfludic-based single-cell integrated proteomic and metabolic assay, we assayed for a panel of signaling, phenotypic, and metabolic regulators at four time points during the first five days of drug treatment. Dimensional reduction of the resultant data set, coupled with information theoretic analysis, uncovered a complex cell state landscape and identified two distinct paths connecting drug-naive and drug-tolerant states. Cells are shown to exclusively traverse one of the two pathways depending on the level of the lineage restricted transcription factor MITF in the drug-naive cells. The two trajectories are associated with distinct signaling and metabolic susceptibilities, and are independently druggable. Our results update the paradigm of adaptive resistance development in an isogenic cell population and offer insight into the design of more effective combination therapies.

/pdf/trajectories-from-snapshots-integrated-proteomic-and-3n485v5gce.pdf

Trajectories from Snapshots: Integrated proteomic and metabolic single-cell assays reveal multiple independent adaptive responses to drug tolerance in a BRAF-mutant melanoma cell line

Production of helper-free retroviruses by transient transfection with a high titer

The ability of tryptophan tRNA (tRNATrP) to initiate reverse transcription of the 70S RNA of avian RNA tumor viruses suggested that the reverse transcriptase (RNAdependent DNA polymerase; deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7) might have a specific binding site for the tRNA. A complex of tRNATrP and the avian myeloblastosis virus reverse transcriptase has been demonstrated using chromatography on Sephadex G-100 columns. Of all the chicken tRNA's, only tRNATrP and a tRNA4Met bind to the enzyme with high enough affinity to be selected from a mixture of the chicken cell tRNAs. The ability of tRNATIP to change the sedimentation rate of the enzyme indicates that tRNATrP is not binding to a contaminant in the enzyme preparation. Treatment of the enzyme with monospecific antibody to reverse transcriptase prevented binding of tRNA as well as inhibited the DNA polymerase activity of the enzyme. The ability of reverse transcriptase to utilize tRNATrP as a primer for DNA synthesis, therefore, appears to involve a highly specific site on the enzyme. In vitro synthesis of DNA by the DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) found in virions of RNA tumor viruses (reverse transcriptase) is a primer-dependent reaction (1, 2). When the "70S" RNA from virions is used as a template, a lowmolecular-weight RNA of the 70S complex acts as primer (3-5). For Rous sarcoma virus, this RNA has been shown to be identical to a cellular tryptophan tRNA (tRNATrP) that is called "spot 1 RNA" after separation by 2-dimensional gel electrophoresis (6-9). Such tRNATrP has been isolated from three sources: 70S RNA, free 4S RNA from virions of Rous sarcoma virus, and normal chicken embryo fibroblasts (4, 5, 8). Although other tRNAs are found associated with 70S RNA and as free 4S RNA in virions (10), 90% of the DNA molecules copied from 70S RNA have been shown to be initiated using tRNATrP as a primer (4). Part of its ability to act as primer may result from the tight association of tRNATrP with the 35S RNA subunits of the 70S RNA; of the 70S-associated tRNAs, tRNATrP requires the highest temperature for removal from the 35S RNA (4, 11). The tRNATrP also appears to be able to anneal back to 35S RNA (12, 13). The association of tRNATrP with 35S RNA, however, is not sufficient to explain its ability to act as primer for the reverse transcriptase because, in general, the enzyme uses DNA efficiently as primer but utilizes RNA primers poorly (1, 14). The apparent selectivity of reverse transcriptase for tRNATrP over other 70S-associated tRNAs and the poor rate of priming of reverse transcriptase by RNA homopolymers led us to investigate whether there might be a specific binding site on the reverse transcriptase for tRNATrP. Abbreviation: AMV, avian myeloblastosis virus., 2535 Using an assay for high-affinity binding sites, we have been able to demonstrate specific binding of tRNATrP to the reverse transcriptase of avian myeloblastosis virus (AMV). MATERIALS AND METHODS Materials. AMV reverse transcriptase was purified by sequential chromatography on columns of DEAE-Sephadex and phosphocellulose (15). The phosphocellulose fraction was further purified on a DNA-cellulose column (16). The enzyme preparation gave only two bands after electrophoretic separation on 10% sodium dodecyl sulfate-polyacrylamide gels; these migrated as do the previously characterized a and ,B subunits of AMV reverse transcriptase (17). Antiserum to AMV reverse transcriptase was obtained from Dr. R. Nowinski, Madison, Wisc. (18). The IgG fraction was purified from serum as described (19). Chicken liver tRNA and chicken liver aminoacyl tRNA synthetases were prepared following published procedures (20-22). Purified Escherichia coli tRNATyr, E. coli tRNASer, and yeast tRNAPhe were gifts from Dr. U. L. RajBhandary and M. Silberklong. (dG)12 18 and (dT)12-18 were purchases from Collaborative Research Corp. These polynucleotides were labeled at their 5'-end using polynucleotide kinase and ['y-32P]ATP (23). Radioactive nucleotides and amino acids were products of New England Nuclear Corp. Sephadex G-100 was obtained from Pharmacia Corp. Preparation of 32P-Labeled RNAs. Cultures of chicken embryo fibroblasts infected with Schmidt-Ruppin D Rous sarcoma virus or Carr-Zilber associated virus were obtained from Dr. H. M. Temin. The cells were radioactively labeled in 100 mm diameter dishes with [32P]orthophosphate as described previously (7), using 1-2 mCi of [32P]orthophosphate per ml of medium. After 48 hr of labeling, virus was harvested from the medium by centrifugation and RNA was extracted without further purification (7). Viral RNA was fractionated into 70S and "free" 4S RNA by centrifugation through sucrose gradients (7). The 4S RNA was precipitated with 2 volumes of ethanol, resuspended in 0.05 M NaCl, 0.01 M Tris-HCI, pH 7.8, 0.01 M MgCl2, and was applied to a 1 ml column of DEAE-Sephadex A-50 (Pharmacia). After successive washes with 0.05 M NaCl and 0.3 M NaCl the RNA was eluted from the column with 1 M NaCl in the same buffer and concentrated by ethanol precipitation. After virus was harvested, the labeled cells were washed twice with 5 ml of TSE buffer (0.02 M Tris-HCI, pH 7.8; 0.1 M NaCl; 1 mM EDTA) at room temperature. Cells were then treated on the culture plates with 0.5% sodium dodecyl sulfate in TSE buffer, 1 ml/10 cm plate for 15 min at room temperature. The suspension was carefully removed from 2536 Biochemistry: Panet et al. the plates to avoid shearing of DNA and 0.25 volume of 5 M NaCl was added. After storage overnight, the precipitate was removed by centrifugation at 15,000 X g for 20 min. The supernatant was extracted twice with phenol and twice with chloroform/isoamyl alcohol (24:1). Nucleic acids were precipitated with 2 volumes of ethanol at -20° in the presence of 100 ,g of carrier RNA (phenol-extracted Sigma type IV yeast RNA). RNA was purified by chromatography on DEAE-Sephadex as described above, followed by centrifugation in a 5-20% sucrose gradient in TSE buffer containing 0.5% Sarkosyl (Geigy) at 49,000 rpm, 2 hr, 40 in a Beckman SW 50.1 rotor. The. individual tRNA species were purified from virionassociated or cellular tRNA using 2-dimensional gel electrophoresis as described previously (7). Unlabeled tRNATrP from chicken liver was purified as described elsewhere (9). E. coli 32P-labeled 5S RNA and 4.5S RNA were prepared as described previously (6) using the hot sodium dodecyl sulfate method of Bremer and Yuan (24). Binding Assay. For assay of binding, the reaction mixture (100-200 ,l) contained 50 mM Tris-HCl (pH 8.3), 60 mM NaCl, 10 mM 2-mercaptoethanol, 10 mM MgCl2, 5% (v/v) glycerol, 0.1% Nonidet P-40, [32P]RNA as indicated, and 50 to 150 enzyme units of reverse transcriptase. After 10 min incubation at 20, the reaction mixtures were chromatographed on columns of Sephadex G-100 (0.6 X 23 cm) preequilibrated at 40 with buffer A (0.1 M potassium phosphate, pH 7.5, 5 mM MgC12, 10 mM 2-mercaptoethanol, 10% (v/v) glycerol, 0.1 mM EDTA, and 0.1% Nonidet P-40). The column was eluted with buffer A. Fractions of 0.17 ml were collected and 25-50 ,ul portions of each fraction were assayed for reverse transcriptase activity; 60-70% of added enzymatic activity was routinely recovered in the fractions. Portions of each of the fractions were also used for determination of [32P]RNA. Enzyme Assay. Reaction mixtures (100 Al) contained 50 mM Tris-HCl (pH 8.3), 6 mM MgCl2, 0.1 mM [3H]dGTP (100 cpm/pmol), 2 Mg of poly(C), 0.5 Mig of (dG)1218, 5 mM dithiothreitol, and 25-50 Ml of fractions from the Sephadex G-100 columns or glycerol gradients. Reactions were incubated for 30 min at 37'. One enzyme unit was defined as the amount needed to catalyze the incorporation of 1 pmol of dGMP into polymer in 1 min. The assay for E. coli DNA polymerase I was as described for reverse transcriptase but 1 Mg of poly(dA) and 1 Mg of (dT)12_18 were used as the template and primer and the incorporation from [3H]dTTP (60 cpm/pmol) was followed.

/pdf/dna-polymerase-reverse-transcriptase-specific-binding-of-ehynexjh9z.pdf

David Baltimore

Papers

Trogocytosis-based cell platform for TCR ligand discovery

Molecular Properties of Voltage-Gated Ion Channels

Trajectories from Snapshots: Integrated proteomic and metabolic single-cell assays reveal multiple independent adaptive responses to drug tolerance in a BRAF-mutant melanoma cell line

Production of helper-free retroviruses by transient transfection with a high titer

DNA Polymerase (Reverse Transcriptase) Specific Binding of Tryptophan Transfer RNA to Avian Myeloblastosis Virus RNA-Dependent