CRISPR-Cas systems have been used with single-guide RNAs for accurate gene disruption and conversion in multiple biological systems. Here we report the use of the endonuclease Cas9 to target genomic sequences in the C. elegans germline, utilizing 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 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 E. coli that can count up to three induction events, the first comprised of a riboregulated transcriptional cascade and the second of 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.A counter is a key component in digital circuits and computing that retains memory of events or objects, representing each number of such as a distinct state. Counters would also be useful in cells, which often must have accurate accounting of tightly controlled processes or biomolecules to effectively maintain metabolism and growth. Counting mechanisms have been reportedly found in telomere length regulation (1,2) and cell aggregation (3). These system behaviors appear to be the result of a thresholding effect in which some critical molecule number or density must be reached for the observed phenotypic change.In this study, we first develop a counter, termed the Riboregulated Transcriptional Cascade (RTC) Counter, which is based on a transcriptional cascade with additional translational regulation. Figs. 1A and 1C illustrate two such cascades that can count up to 2 and 3, respectively (hence, the designations RTC 2-Counter and RTC 3-Counter). For the RTC 2-Counter, the constitutive promoter P Ltet0-1 drives transcription of T7 RNA polymerase (RNAP), whose protein binds the T7 promoter and transcribes the downstream gene, in this case Green Fluorescent Protein (GFP). Both genes are additionally regulated by riboregulators (4), whose cis and trans elements silence and activate post-transcriptional gene expression, respectively. The cis-repressor sequence (cr) is placed between the transcription start site and the ribosome binding site (RBS), and its complementarity with the RBS causes a stem-loop structure to form upon transcription. This secondary structure prevents binding of the 30S ribosomal subunit to the RBS, inhibiting translation. A short, trans-activating, noncoding RNA (taRNA) driven by the arabinose promoter P BAD binds to the cis-repressor in trans, relieving * This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the Copyright Act without the prior, written permission of AAAS. * These authors contributed equally to this work. 1A). With cis-repressed T7 RNAP mRNAs in the cell, the first pulse of arabinose drives a short burst of taRNA production and consequently expression of...

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BackgroundCRISPR-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.ResultsWe 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.ConclusionOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0817-8) contains supplementary material, which is available to authorized users.

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.

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.

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.

24We report a high-throughput screening approach to measure Staphylococcus aureus 25 Cas9 (SaCas9) genome editing variation in human cells across a large repertoire of 26 88,692 single guide RNAs (sgRNAs) paired with matched or mismatched target sites in 27 a synthetic cassette. We incorporated randomized barcodes that enable 'whitelisting' of 28 correctly synthesized molecules for further downstream analysis, in order to circumvent 29 the limitation of oligonucleotide synthesis errors. We find SaCas9 sgRNAs with a 21-30 nucleotide spacer are most active against off-targets with single and double mismatches, 31 compared to shorter or longer sgRNAs. Using this dataset, we developed an SaCas9 32 specificity model that performs well in ranking off-target sites. The barcoded pairwise 33 library screen enabled high-fidelity recovery of guide-target relationships, providing a 34 peer-reviewed)