COMMISSIONING OF THE FERMILAB ELECTRON COOLER PROTOTYPE BEAM LINE

Abstract A prototype of a 4.3-MeV electron cooling system is being assembled at Fermilab as part of the ongoing R&D program in high energy electron cooling. This electron cooler prototype will not demonstrate the actual cooling but it will allow determining if the electron beam properties are suitable for antiproton beam cooling. An electron beam is accelerated by a 5-MV Pelletron (Van de Graaff type) accelerator and transported to a prototype cooling section. The cooling will take place in a 20-m long solenoid flanked on both sides by a delivery and return beam-line -a total of 60 meters of transport channel. This paper describes the first results of commissioning this novel beam line as well as the status of the electron cooling R&D program

RNA targeting with CRISPR–Cas13

RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference1-3 can efficiently knockdown RNAs, but it is prone to off-target effects4, and visualizing RNAs typically relies on the introduction of exogenous tags5. Here we demonstrate that the class 2 type VI6,7 RNA-guided RNA-targeting CRISPR-Cas effector Cas13a8(previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049

A Natural Love of Natural Products

Recent research on the chemistry of natural products from the author’s group that led to the receipt of the ACS Ernest Guenther Award in the Chemistry of Natural Products is reviewed. REDOR NMR and synthetic studies established the T-taxol conformation as the bioactive tubulin-binding conformation, and these results were confirmed by the synthesis of compounds which clearly owed their activity or lack of activity to whether or not they could adopt the T-taxol conformation. Similar studies with the epothilones suggest that the current tubulin-binding model needs to be modified. Examples of natural products discovery and biodiversity conservation in Suriname and Madagascar are also presented, and it is concluded that natural products chemistry will continue to make significant contributions to drug discovery. My first real exposure to natural products chemistry came in my third and final year as an undergraduate at Cambridge University, when I attended a course of lectures on the chemistry of natural products by the Nobel Prize-winning chemist Sir Alexander Todd (later to become Lord Todd). The lectures included many references to his own work in the field, with stories of his early work on the structure of cholesterol, th

CRISPR-Cas orthologues and variants: optimizing the repertoire, specificity and delivery of genome engineering tools

Robust and cost-effective genome editing in a diverse array of cells and model organisms is now possible thanks to the discovery of the RNA-guided endonucleases of the CRISPR-Cas system. The commonly used Cas9 of Streptococcus pyogenes shows high levels of activity but, depending on the application, has been associated with some shortcomings. Firstly, the enzyme has been shown to cause mutagenesis at genomic sequences resembling the target sequence. Secondly, the stringent requirement for a specific motif adjacent to the selected target site can limit the target range of this enzyme. Lastly, the physical size of Cas9 challenges the efficient delivery of genomic engineering tools based on this enzyme as viral particles for potential therapeutic applications. Related and parallel strategies have been employed to address these issues. Taking advantage of the wealth of structural information that is becoming available for CRISPR-Cas effector proteins, Cas9 has been redesigned by mutagenizing key residues contributing to activity and target recognition. The protein has also been shortened and redesigned into component subunits in an attempt to facilitate its efficient delivery. Furthermore, the CRISPR-Cas toolbox has been expanded by exploring the properties of Cas9 orthologues and other related effector proteins from diverse bacterial species, some of which exhibit different target site specificities and reduced molecular size. It is hoped that the improvements in accuracy, target range and efficiency of delivery will facilitate the therapeutic application of these site-specific nucleases

Professional Precision Laser Inclinometer: the Noises Origin and Signal Processing

In the Professional Precision Laser Inclinometer the noise origins were analyzed. The new efficient methodic of these noises taking into account was proposed and realized. The own (instrumental) noise 6 × 10$^{–12}$ rad/Hz$^{1/2}$ of the PPLI has been measured in the frequency range of [0.1 Hz, 1 Hz]. The minimal PPLI registered spectral density when analyzing of a daylong data was 2.4 × 10$^{–11}$ rad/Hz$^{1/2}$

The Seismic Angular Noise of an Industrial Origin Measured by the Precision Laser Inclinometer in the LHC Location Area

The decreasing of the relaxation time for recovery of the horizontality of the surface of thin layer of the liquid in the cuvette is a new observed phenomenon caused by the meniscus influence in case of the cuvette small inclination. It was experimentally established that the time of reconstruction of the surface of 4 mm thick liquid layer in the cuvette of ∅5 mm at 0.5 µrad of calibration inclining is 0.082 ± 0.006 s. In this case the relative deviation from the liquid surface planarity does not exceed 7%. The physics explanation of the phenomenon observed is proposed. The taking into account of the time duration of liquid surface horizontality reconstruction in the Precision Laser Inclinometer has resulted in the widening of Inclinometer’s sensitivity range up to 12.3 ± 0.9 Hz. The data obtained could be used when seismoisolating of the research equipment

Compact Precision Laser Inclinometer: Measurement of Signals and Noise

The results of developing a compact version of the Precision Laser Inclinometer (CPLI) with the reduced overall dimensions of 20 × 20 × 20 cm and weight of 10 kg are presented. Experimental data on detected angular oscillations of the Earth’s surface at the JINR site are obtained. The achieved sensitivity is 6 × 10$^{–11}$ rad/Hz$^{1/2}$ in the frequency range 1.4 × 10$^{–3}$–10 Hz. The CPLI can be used in modern physical experiments for seismic isolation of large-scale installations. Reduction of the impact of microseismic angular oscillations of the Earth’s surface on the sensitive elements of the VIRGO Interference Gravitational Antenna, the Large Hadron Collider, and NICA will increase the accuracy of the experiments

Programmable RNA recognition and cleavage by CRISPR/Cas9

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA:DNA complementarity to identify target sites for sequence-specific doublestranded DNA (dsDNA) cleavage(1-5). In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, the protospacer adjacent motif (PAM), next to and on the strand opposite the 20-nucleotide target site in dsDNA(4-7). Cas9 has proven to be a versatile tool for genome engineering and gene regulation in many cell types and organisms(8), but it has been thought to be incapable of targeting RNA(5). Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalyzed DNA cleavage(7). Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous mRNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable and tagless transcript recognition