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

Stepwise Formation of Proteins on Ribosomes

We have constructed an 8-base-pair insertion mutation in the 3' noncoding region of an infectious poliovirus cDNA clone that gives rise to a temperature-sensitive RNA synthesis mutant upon transfection into mammalian cells. The mutated cDNA was used to establish a cell line that releases the mutant poliovirus in a temperature-dependent fashion, repre- senting a unique persistent viral infection. A poliovirus mutant mapping in the noncapsid region of the viral genome can be complemented in this cell line, implying that the cell line expresses viral proteins at the nonpermissive temperature. Many events take place in a cell infected by poliovirus. The positive-strand RNA genome of the virus must be replicated, viral proteins made, cellular functions inhibited, and new virions produced. Although much has been learned about these and other aspects of the virus life cycle (1), clear understanding of the functions of the various viral proteins and their interaction with cellular constituents still lies ahead. This is due in part to the lack of well-defined poliovirus mutants. The discovery that a cloned cDNA copy of the poliovirus RNA genome gives rise to infectious poliovirus upon transfection into mammalian cells (2, 3) opened up the possibility of constructing defined poliovirus mutants by mutagenizing the infectious cDNA. The opportunity to store the genetic information encoding a mutant poliovirus as a cDNA clone makes it possible to determine the nucleotide sequence of the mutated genome and to ensure that the mutation is responsible for the observed phenotype. We report here the construction of several linker-insertion mutations in the 3' noncoding region of the viral genome. One of these mutations, PTH7387, gave rise to a temperature- sensitive poliovirus mutant. The infectious cDNA bearing this mutation was used to establish stable persistently infect- ed human HeLa cell lines that release infectious poliovirus at the permissive temperature. Such cell lines, at the non- permissive temperature, can complement another poliovirus mutant, showing that the integrated viral genome is translat- ed.

A poliovirus temperature-sensitive RNA synthesis mutant located in a noncoding region of the genome (poliovirus cDNA/site-directed mutagenesis/persistent viral infection/complementation)

T Cell Immunotherapy: Optimizing Trial Design

Edward J. Siden and David Baltimore Department of Biology and Center for Cancer Research Massachusetts Institute of Technology Cambridge, Massachusetts 02139 Daniel Clark and Naomi E. Rosenberg Department of Pathology and Cancer Research Center Tufts University School of Medicine Boston, Massachusetts 02111 Summary The majority of cell lines derived by infection of murine bone marrow cells with Abelson murine leukemia virus (A-MuLV) synthesize a p chain but no detectable light chain. Aside from this p-only phenotype, lines that make only light chain, both chains or no immunoglobulin-related polypep- tides have also been found. Two lines have been studied in detail: one that makes only p chain and one that makes only

Synthesis by Lymphoid Cells Transformed in Vitro by Abelson Murine Leukemia Virus

According to some embodiments herein, expression systems and methods for reversible gene expression are provided. In some embodiments, adeno-associated viral vectors encoding a gene product of interest and comprising a plurality of recombinase target sites are provided. In some embodiments, a source of recombinase is provided. In some embodiments, the gene product of interest is expressed, and the recombinase then induces recombination events between the recombinase target sites, thus reducing or eliminating expression of the gene product of interest.

David Baltimore

Papers

Stepwise Formation of Proteins on Ribosomes

A poliovirus temperature-sensitive RNA synthesis mutant located in a noncoding region of the genome (poliovirus cDNA/site-directed mutagenesis/persistent viral infection/complementation)

T Cell Immunotherapy: Optimizing Trial Design

Synthesis by Lymphoid Cells Transformed in Vitro by Abelson Murine Leukemia Virus

Reversible gene expression