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

Thegene encoding interleukin-2 (IL-2) contains a sequence52to326nucleotides upstreamofits transcriptional initiation site thatpromotes transcription inTcells thathavebeenactivated bycostimulation withtetradecanoyl phorbol myristyl acetate (TPA)andphytohemagglutinin (PHA).We foundthatthe ubiquitous transcription factor, Oct-1, boundtotwopreviously identified motifs within thehumanIL-2 enhancer, centered atnucleotides -74and-251.Eachsite intheIL-2enhancer thatboundOct-1 invitro was also required toachieve amaximal transcriptional responsetoTPAplusPHA invivo. Point mutations within either theproximal ordistal octamer sequencesreduced theresponseoftheenhancer toactivation by54and 34%,respectively. Because themurine T-cell line EL4constitutively expressesOct-2 andrequires onlyTPAto induce transcription oftheIL-2 gene,theeffect ofOct-2 expression onactivation oftheIL-2promoter inJurkat Tcellswas determined. Expression ofOct-2 potentiated transcription 13-fold inresponsetoTPAplusPHAand permited theenhancer torespond tothesingle stimulus ofTPA.Therefore, boththesignal requirements and themagnitude ofthetranscription responseoftheIL-2promoter can bemodulated byOct-2.

ThePromoter oftheHumanInterleukin-2 GeneContains Two Octamer-Binding Sites andIsPartially Activated bythe Expression ofOct-2

Verwendung chimerer nukleasen zur stimulierung des gen-targeting

What Are the Implications for Training at the Doctoral Level

TheLongTerminal RepeatIsNota MajorDeterminant ofthe Cellular Tropism ofHumanImmunodeficiency VirusType1

Dans le procede intracellulaire decrit, qui sert a inhiber l'HIV (virus de l'immunodeficience humaine) dans des cellules de mammiferes, une structure recombinante est introduite dans les cellules. La structure recombinante comprend: (a) la chaine de repetition de terminaison longue (LTR) de l'HIV ou une partie de la LTR d'HIV, qui contient un promoteur d'HIV fonctionnel et un ADN d'origine non HIV codant pour un produit qui est toxique aux cellules infectees par l'HIV, lorsqu'il est present dans ces cellules seul ou en conjonction avec une substance selectionnee; ou (b) la totalite de la LTR d'HIV ou une partie de la LTR d'HIV, qui contient un promoteur d'HIV fonctionnel et un ADN d'HIV ayant subit une mutation ou une alteration. Des compositions servant a inhiber l'HIV, qui contiennent de telles structures recombinantes, sont egalement decrites.

David Baltimore

Papers

ThePromoter oftheHumanInterleukin-2 GeneContains Two Octamer-Binding Sites andIsPartially Activated bythe Expression ofOct-2

Verwendung chimerer nukleasen zur stimulierung des gen-targeting

What Are the Implications for Training at the Doctoral Level

TheLongTerminal RepeatIsNota MajorDeterminant ofthe Cellular Tropism ofHumanImmunodeficiency VirusType1

Procede intracellulaire d'inhibition de l'hiv dans des cellules de mammiferes