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.

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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

A radioimmunoassay was developed that can detect and quantitate 3 ng or more of the avian RNA tumor virus reverse transcriptase. The assay detected no antigenic sites in Rous sarcoma virus alpha virions or in virions of a murine RNA tumor virus. About 70 molecules of reverse transcriptase were found per virion of avian myleloblastosis virus with this assay or with an assay based on antibody inhibition of enzymatic activity. The assay detected about 270 ng of enzyme per mg of cell protein in virus-producing cells; uninfected cells had much less antigenic material but contained some determinants able to displace radioactive antigen. No additional antigenic determinants on reverse transcriptase could be detected that were not found on the separated alpha subunit of the enzyme. Although sevenfold less sensitive than enzymatic activity as a measure of reverse transcriptase, the radioimmunoassay can detect antigen using small amounts of protein and in the presence of inhibtors.

Quantitation of avian RNA tumor virus reverse transcriptase by radioimmunoassay.

RelB-deficient mice ( relB− /−) have a complex phenotype including multiorgan inflammation and hematopoietic abnormalities. To examine whether other NF-κB/Rel family members are required for the development of this phenotype or have a compensatory role, we have initiated a program to generate double-mutant mice that are deficient in more than one family member. Here we report the phenotypic changes in relB− /− mice that also lack the p50 subunit of NFκB ( p50− /−). The inflammatory phenotype of p50− /− relB− /− double-mutant mice was markedly increased in both severity and extent of organ involvement, leading to premature death within three to four weeks after birth. Double-knockout mice also had strongly increased myeloid hyperplasia and thymic atrophy. Moreover, B cell development was impaired and, in contrast to relB− /− single knockouts, B cells were absent from inflammatory infiltrates. Both p50− /− and heterozygous relB− /+ animals are disease-free. In the absence of the p50, however, relB− /+ mice ( p50− /− relB −/+) had a mild inflammatory phenotype and moderate myeloid hyperplasia. Neither elevated mRNA levels of other family members, nor increased κB-binding activities of NF-κB/Rel complexes could be detected in single- or double-mutant mice compared to control animals. These results indicate that the lack of RelB is, in part, compensated by other p50-containing complexes and that the “classical” p50-RelA–NF-κB activity is not required for the development of the inflammatory phenotype.

p50–NF-κB Complexes Partially Compensate for the Absence of RelB: Severely Increased Pathology in p50−/−relB−/−Double-knockout Mice

A new ribonucleic acid polymerase appearing after mengovirus infection of l-cells.

The invention provides systems and methods for the generation of lymphocytes having a unique antigen specificity. In a preferred embodiment, the invention provides methods of virally infecting cells from bone marrow with one or more viral vectors that encode antigen-specific T cell receptors. The resulting lymphocytes, and in particular, T cells express the T cell receptor (TCR) that was introduced. The lymphocytes generated can be used for a variety of therapeutic purposes including the treatment of various cancers and the generation of a desired immune response to viruses and other pathogens. The resulting cells develop normally and respond to antigen both in vitro and in vivo. It is possible to modify the function of lymphocytes by using stem cells from different genetic backgrounds. Thus the system constitutes a powerful tool to generate desired lymphocyte populations both for research and therapy. Applications of this technology include treatments for infectious diseases, such as HIV/AIDS, cancer therapy, allergy, and autoimmune disease.

Method for the generation of antigen-specific lymphocytes

The pairwise complementarity of the nucleotide residues in nucleic acids provides a simple mechanism for the specification of the sequence of residues in a nucleic acid. A molecule with a given sequence will specify a molecule with a complementary sequence and, in turn, the complementary molecule can specify the sequence of the original one. In the case of single-stranded RNA viruses, it is thus likely that a strand of complementary RNA will be the intracellular template for the synthesis of viral RNA (the molecule which exists in mature particles). This argument is strengthened by the finding of double-stranded RNA in cells infected with single-stranded RNA viruses. The hypothesis that the complementary molecule of RNA, in a double-stranded RNA, is the template for viral RNA synthesis requires that there be a region of hydrogen bonding between the growing molecule of viral RNA and the complementary RNA. As the new chain of viral RNA elongates, either it displaces the viral strand of the double-stranded RNA or it is not hydrogen-bonded except in proximity to the growing point. In either case, the growing strand of viral RNA plus its template will form a complex which will be partially single- and partially double-stranded. Since more than one molecule of nascent viral RNA could be attached to one double-stranded molecule, such complexes might be fairly large and easily distinguishable from true double-stranded RNA by their content of single-stranded RNA.
We shall present evidence in this paper for the existence of such a complex of single- and double-stranded RNA in cells infected with poliovirus. It is similar to a structure first identified in bacteria infected with an RNA bacteriophage which was named the replicative intermediate (RI). We shall retain this nomenclature.

David Baltimore

Papers

Quantitation of avian RNA tumor virus reverse transcriptase by radioimmunoassay.

p50–NF-κB Complexes Partially Compensate for the Absence of RelB: Severely Increased Pathology in p50−/−relB−/−Double-knockout Mice

A new ribonucleic acid polymerase appearing after mengovirus infection of l-cells.

Method for the generation of antigen-specific lymphocytes

An Intermediate in the Synthesis of Poliovirus RNA.