To understand the molecular processes underlying aging, we screened modENCODE ChIP-seq data to identify transcription factors that bind to age-regulated genes in C. elegans. The most significant hit ...was the GATA transcription factor encoded by elt-2, which is responsible for inducing expression of intestinal genes during embryogenesis. Expression of ELT-2 decreases during aging, beginning in middle age. We identified genes regulated by ELT-2 in the intestine during embryogenesis, and then showed that these developmental genes markedly decrease in expression as worms grow old. Overexpression of elt-2 extends lifespan and slows the rate of gene expression changes that occur during normal aging. Thus, our results identify the developmental regulator ELT-2 as a major driver of normal aging in C. elegans.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
CRISPR-Cas systems have been broadly embraced as effective tools for genome engineering applications, with most studies to date utilizing the Streptococcus pyogenes Cas9. Here we characterize and ...manipulate the smaller, 1053 amino acid nuclease Staphylococcus aureus Cas9.
We find that the S. aureus Cas9 recognizes an NNGRRT protospacer adjacent motif (PAM) and cleaves target DNA at high efficiency with a variety of guide RNA (gRNA) spacer lengths. When directed against genomic targets with mutually permissive NGGRRT PAMs, the S. pyogenes Cas9 and S. aureus Cas9 yield indels at comparable rates. We additionally show D10A and N580A paired nickase activity with S. aureus Cas9, and we further package it with two gRNAs in a single functional adeno-associated virus (AAV) vector. Finally, we assess comparative S. pyogenes and S. aureus Cas9 specificity using GUIDE-seq.
Our results reveal an S. aureus Cas9 that is effective for a variety of genome engineering purposes, including paired nickase approaches and all-in-one delivery of Cas9 and multiple gRNA expression cassettes with AAV vectors.
We report the use of clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease Cas9 to target genomic sequences in the Caenorhabditis elegans germ line using ...single-guide RNAs that are expressed from a U6 small nuclear RNA promoter. Our results demonstrate that targeted, heritable genetic alterations can be achieved in C. elegans, providing a convenient and effective approach for generating loss-of-function mutants.
Synthetic Gene Networks That Count Friedland, Ari E; Lu, Timothy K; Wang, Xiao ...
Science (American Association for the Advancement of Science),
05/2009, Letnik:
324, Številka:
5931
Journal Article
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Synthetic gene networks can be constructed to emulate digital circuits and devices, giving one the ability to program and design cells with some of the principles of modern computing, such as ...counting. A cellular counter would enable complex synthetic programming and a variety of biotechnology applications. Here, we report two complementary synthetic genetic counters in Escherichia coli that can count up to three induction events: the first, a riboregulated transcriptional cascade, and the second, a recombinase-based cascade of memory units. These modular devices permit counting of varied user-defined inputs over a range of frequencies and can be expanded to count higher numbers.
We adapted the CRISPR-Cas9 system for template-mediated repair of targeted double-strand breaks via homologous recombination in Caenorhabditis elegans, enabling customized and efficient genome ...editing. This system can be used to create specific insertions, deletions, and base pair changes in the germline of C. elegans.
Therapeutic genome editing with Staphylococcus aureus Cas9 (SaCas9) requires a rigorous understanding of its potential off-target activity in the human genome. Here we report a high-throughput ...screening approach to measure SaCas9 genome editing variation in human cells across a large repertoire of 88,692 single guide RNAs (sgRNAs) paired with matched or mismatched target sites in a synthetic cassette. We incorporate randomized barcodes that enable whitelisting of correctly synthesized molecules for further downstream analysis, in order to circumvent the limitation of oligonucleotide synthesis errors. We find SaCas9 sgRNAs with 21-mer or 22-mer spacer sequences are generally more active, although high efficiency 20-mer spacers are markedly less tolerant of mismatches. Using this dataset, we developed an SaCas9 specificity model that performs robustly in ranking off-target sites. The barcoded pairwise library screen enabled high-fidelity recovery of guide-target relationships, providing a scalable framework for the investigation of CRISPR enzyme properties and general nucleic acid interactions.
The original HTML version of this Article incorrectly listed an affiliation of Josh Tycko as 'Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA', instead of the ...correct 'Present address: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA'. It also incorrectly listed an affiliation of this author as 'Present address: Arrakis Therapeutics, 35 Gatehouse Dr., Waltham, MA, 02451, USA'.The original HTML version incorrectly listed an affiliation of Luis A. Barrera as 'Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 06511, USA', instead of the correct 'Present address: Arrakis Therapeutics, 35 Gatehouse Dr., Waltham, MA 02451, USA'.Finally, the original HTML version incorrectly omitted an affiliation of Nicholas C. Huston: 'Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, USA'.This has been corrected in the HTML version of the Article. The PDF version was correct from the time of publication.
The clustered, regularly interspaced, short, palindromic repeat (CRISPR)-associated (CAS) nuclease Cas9 has been used in many organisms to generate specific mutations and transgene insertions. Here ...we describe a method using the S. pyogenes Cas9 in C. elegans that provides a convenient and effective approach for making heritable changes to the worm genome.
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