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

Inflammation involves timed gene expression, suggesting that the fine-tuned onset, amplitude, and termination of expression of hundreds of genes is of critical importance to organismal homeostasis. Recent study of post-transcriptional regulation of inflammatory gene expression led to the suggestion of a regulatory role for pre-mRNA splicing. Here, using a hybrid capture approach to purify incompletely spliced, chromatin-associated pre-mRNAs, we use deep sequencing to study pre-mRNA splicing of the NF-kB transcriptome. By freezing transcription and examining subsequent splicing of complete transcripts, we find many introns splice tens to hundreds of times slower than average. In many cases, this is attributable to poor splice donor sequences that are evolutionarily conserved. When these introns were altered by ~2 base pairs to yield stronger splice donors, gene expression levels increased markedly for several genes in the context of a reporter system. We propose that such splice sites represent a regulatory mechanism that determines the timing of production of the mRNAs from certain inflammatory genes and may also limit mRNA expression from these genes. Further work will be needed to understand the roles of this regulation in the inflammatory response. The suggestion of extensive temporal regulation of pre-mRNA splicing as a regulatory process in inflammation raises the question of where else in biology there may be timed processes with a similar underlying cause.

/pdf/programmed-delayed-splicing-a-mechanism-for-timed-4l7kyiwmgt.pdf

Programmed Delayed Splicing: A Mechanism for Timed Inflammatory Gene Expression

Tumor neoantigens are fragments of mutated proteins that contain the mutation, and can be presented by major histocompatibility complex molecules on tumor cells, where they are surveyed by T cells. The rapid and sensitive identification of neoantigen‐specific T cell populations from tumor tissues or blood has proven challenging. A microchip platform for the non‐destructive identification of neoantigen‐specific CD8 T cells is described. The method utilizes a library of neoantigen/MHC tetramers linked to a magnetic nanoparticle via a DNA barcode. The neoantigen‐specificity of the T cells is determined by decoding the barcode through sequential fluorescent microscopy reads. The captured T cells may be further characterized for function, or via matching the neoantigen‐specificity with the T cell receptor gene. Tumor infiltrating lymphocytes and non‐expanded peripheral blood mononuclear cells collected from melanoma patients at various time points across an anti‐PD1 therapy regimen are shown to contain overlapping neoantigen‐specific T cell populations.

Sensitive, Non‐Destructive Detection and Analysis of Neoantigen‐Specific T Cell Populations from Tumors and Blood

Substitutions intheProtease (3CPrO) GeneofPoliovirus Can Suppress a Mutation inthe5'Noncoding Region

We used mouse-Chinese hamster somatic cell hybrids which lose mouse chromosomes to examine the distribution of murine leukemia virus DNA sequences in the genome of A/HeJ mice. We analyzed total cellular DNA from various hybrid clones for the presence of viral sequences by molecular hybridization and used the Southern blot hybridization procedure to identify viral DNA in cellular restriction endonuclease fragments. Our results show that murine leukemia virus DNA sequences are distributed among many mouse chromosomes in this strain. Chromosome 4 was shown to contain murine leukemia virus DNA sequences.

Distribution of endogenous murine leukemia virus DNA sequences among mouse chromosomes

There is sufficient evidence that the A-MuLV protein is a tyrosine-specific protein kinase. There are methods for detecting this kinase activity and this kinase has been expressed in E. coli. Because the information coding for the tyrosine-specific protein kinase is present in normal mouse cells, such an enzyme must have a normal physiologic function. The elucidation of this physiologic function and the understanding of the role of this enzyme activity in neoplastic transformation is the challenge of the future.

David Baltimore

Papers

Programmed Delayed Splicing: A Mechanism for Timed Inflammatory Gene Expression

Sensitive, Non‐Destructive Detection and Analysis of Neoantigen‐Specific T Cell Populations from Tumors and Blood

Substitutions intheProtease (3CPrO) GeneofPoliovirus Can Suppress a Mutation inthe5'Noncoding Region

Distribution of endogenous murine leukemia virus DNA sequences among mouse chromosomes

[40] Characterization of the abelson murine leukemia virus-encoded tyrosine-specific protein kinase