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

Letter to the editorRetraction: Altered repertoire of endogenous immunoglobulin gene expression in transgenic mice containing a rearranged Mu heavy chain gene

Provided are methods for generating immune cells of the desired type and specificity in a host. The methods may be used to treat a disease or disorder, such as a tumor, in a patient (Figure 6). Target cells, preferably hematopoietic stem cells such as primary bone marrow cells are transfected with a polynucleotide encoding a T cell receptor with the desired specificity. The transfected cells are then transferred to the host where they develop into mature, functional immune cells. The source of the T cell receptor can determine the stem cell's fate. Thus transfecting stem cells with TCRs from cytotoxic cells will lead to the generation of cytotoxic T cells in the host, while TCRs from helper cells will produce helper cells. Both arms of T cell immunity can be generated simultaneously in a host. Additionally, the immune response to the desired antigen can be further stimulated by immunizing the host with the antigen.

Antigen specific t cell therapy

A TLR4 agonist synergizes with dendritic cell-directed lentiviral vectors for inducing antigen-specific immune responses.

How biology became an information science

The v-abl and v-src oncogenes encode protein-tyrosine kinases that possess different biological properties in spite of their high degree of amino acid conservation. To correlate functional differences with structural domains of the two oncogenes, we recombined v-abl and v-src just downstream of the lysines in their ATP-binding sites, within the kinase domain. The biological activity of the chimeric genes was studied and compared with that of v-src and v-abl. The v-src/v-abl recombinant shared with v-src and v-abl the ability to transform fibroblasts. In addition, like v-abl, it transformed lymphoid cells and relieved a hematopoietic cell line of its interleukin 3 requirement. In contrast, the reciprocal construct, v-abl/v-src, was transformation defective. Lack of biological activity correlated with formation of a stable complex between the chimeric protein and two cellular proteins and with low kinase activity. We conclude that the specificity within the kinase domain determines the particular biological behavior of protein-tyrosine kinase oncogenes.

/pdf/recombinants-within-the-tyrosine-kinase-region-of-v-abl-and-6aj9e0zf2j.pdf

David Baltimore

Papers

Letter to the editorRetraction: Altered repertoire of endogenous immunoglobulin gene expression in transgenic mice containing a rearranged Mu heavy chain gene

Antigen specific t cell therapy

A TLR4 agonist synergizes with dendritic cell-directed lentiviral vectors for inducing antigen-specific immune responses.

How biology became an information science

Recombinants within the tyrosine kinase region of v-abl and v-src identify a v-abl segment that confers lymphoid specificity.