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

Methods for producing HAV cDNA, products thereof, and uses thereof. HAV cDNA is produced, for example, by reverse transcribing HAV RNA and subsequently inserting the HAV cDNA into bacterial plasmids by genetic-engineering techniques. Transformed bacteria are then cloned and cultured to produce replicated chimeric plasmids containing the HAV cDNA. Such HAV cDNA is useful in assaying for the presence of HAV and in the production of HAV antigen and in the production of antibodies against HAV.

PRODUCTION OF cDNA REPRESENTING HEPATITIS A VIRAL SEQUENCES

In vitro replication of rna tumor viruses

The content and size of the poly(A) on Mengovirus RNA grown in both mouse L cells and HeLa cells have been examined. Virion RNA from either cell line could bind to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. The size of the poly(A) on the Mengovirus RNA was independent of the host cell and averaged from 50 to 70 nucleotides.

/pdf/poly-a-on-mengovirus-rna-25nmaw3d6v.pdf

Poly(A) on Mengovirus RNA

Terminal deoxynucleotidyl transferase in akr leukemic cells and lack of relation of enzyme activity to cell cycle phase.

S oon the Bush administration will decide the fate of human embryonic stem cell (ESC) research at U.S. government-funded institutions, and the outcome of that decision will greatly influence the role of ESC science in human developmental biology around the world. But although the forces that science brings to this field are powerful, the future of ESC research will largely be determined by other interests: politics, organized religion, commerce, the legal community, and patient advocacy groups. The decision-making process needs to develop a policy that is fact-based and serves the best interests of society as well as science.

Broadly, stem cells are rare cells that renew themselves and, in addition, give rise to differentiated cells. Some, from adult tissues, have multiple but usually restricted developmental potency: Hematopoietic stem cells (HSCs), for example, normally make only blood cells; multipotent brain stem cells make only brain cells. ESCs, in contrast, are derived from cells isolated from the inner cavity of the blastocyst: an early embryo that cannot develop further unless it successfully implants in the uterus. These cells are pluripotent: Given an encouraging environment, they can develop into any cell type; although so far in culture they have been unable to form whole organs, much less bodies.

It would be convenient if pluripotent cells persisted into adulthood. Unfortunately, most (but not all) published accounts suggesting adult stem cell pluripotency have not successfully established that one type can produce a cell of another tissue type. Indeed, although HSCs capable of regenerating the blood can be isolated from adults or fetuses, so far brain stem cells capable of robust growth and transplantability have come only from fetal or ESC sources. This is likely to be true for a number of tissues; fetal stem cells are much more active than postneonatal cells. A moratorium on research and/or transplantation of fetal stem cells could thus be devastating. As for the search for pluripotent adult stem cells, it is always possible, perhaps even likely, that further research might reveal a source. But that is simply a hope, and it would be foolish to abandon the surer path for the unproven one.

The greatest mysteries of human development lie in the time interval between the early blastocyst embryo and the much later point at which organs are formed. What is needed is a thorough understanding of the cellular and genetic events that make the organs and tissues from these pluripotent cells. That can come only from a broad-scale attack on the problem from all kinds of researchers, whether in academic, nonprofit, or commercial organizations. The outcome will surely have profound effects on medicine, science, and translational research. Yet these advances that could be gained from human ESC research are currently restricted in the United States to the single commercial entity that holds the patents for the ESCs developed using its own funding. That is a disturbing anomaly, but it can be remedied by a decision to approve ESC research by government-funded entities.

Decisions to approve ESC research have been recently undertaken in several other countries, notably Germany and the United Kingdom, where government sought advice from a broad range of concerned interests. Not only does that provide a constructive example to our administration, it underscores another consequence of the wrong decisions here. Failure to make this research opportunity available in the United States will make those countries centers of the scientific, medical, and commercial advances in which we ought to be a contributing partner.

Plainly, scientists alone should not make the decisions about the ethical conduct of their work or about its social applications. It is appropriate that governments, with appropriate public input, define the societal interest in particular lines of research. But in making those policies, the state should minimize purely political considerations and be mindful of the separation of church and state. The wrong action here could close the door to an important avenue of scientific and clinical discovery. The state should not be the barrier to the translation of these potentially revolutionary therapeutic opportunities into real medical advances.

David Baltimore

Papers

PRODUCTION OF cDNA REPRESENTING HEPATITIS A VIRAL SEQUENCES

In vitro replication of rna tumor viruses

Poly(A) on Mengovirus RNA

Terminal deoxynucleotidyl transferase in akr leukemic cells and lack of relation of enzyme activity to cell cycle phase.

Disappearing stem cells, disappearing science.