A continuous evolution system for contracting the host range of bacteriophage T7.

Bacteriophage T7 is an intracellular parasite that recognizes its host via its tail and tail fiber proteins, known as receptor-binding proteins (RBPs). The RBPs attach to specific lipopolysaccharide (LPS) features on the host. Various studies have shown expansion of the phage's host range via mutations in the genes encoding the RBPs, whereas only a few have shown contraction of its host range. Furthermore, most experimental systems have not monitored the alteration of host range in the presence of several hosts simultaneously. Here we show that T7 phage grown in the presence of five restrictive strains and one permissive host, each with a different LPS form, gradually avoids recognition of the restrictive strains. Remarkably, avoidance of the restrictive strains was repeated in different experiments using six different permissive hosts. The evolved phages carried mutations that changed their specificity, as determined by sequencing of the genes encoding the RBPs. This system demonstrates a major role for RBPs in narrowing the range of futile infections. The system can be harnessed for host-range contraction in applications such as detection or elimination of a specific bacterial serotype by bacteriophages.

A genetic system for biasing the sex ratio in mice.

Biasing the sex ratio of populations of different organisms, including plants, insects, crustacean, and fish, has been demonstrated by genetic and non-genetic approaches. However, biasing the sex ratio of mammalian populations has not been demonstrated genetically. Here, we provide a first proof of concept for such a genetic system in mammals by crossing two genetically engineered mouse lines. The maternal line encodes a functional Cas9 protein on an autosomal chromosome, whereas the paternal line encodes guide RNAs on the Y chromosome targeting vital mouse genes. After fertilization, the presence of both the Y-encoded guide RNAs from the paternal sperm and the Cas9 protein from the maternal egg targets the vital genes in males. We show that these genes are specifically targeted in males and that this breeding consequently self-destructs solely males. Our results pave the way for a genetic system that allows biased sex production of livestock.

Optimizing DNA transduction by selection of mutations that evade bacterial defense systems.

We recently developed a platform where phage-transducing particles optimize DNA delivery to a wide range of hosts. Here, we use this platform to optimize DNA transduction into hosts that naturally restrict specific DNA sequences. We first show that a specific plasmid is restricted for transduction into a particular Salmonella strain. Using the platform, we select for a mutated plasmid that overcomes the restriction barrier. Insertion of the non-mutated sequence into a permissive plasmid restricts transduction. We further show that epigenetic modification enables the DNA to evade restriction by the putative defense system. Our results validate this straightforward genetic approach for optimization of DNA transduction into new hosts.

A technological and regulatory outlook on CRISPR crop editing.

Generating plants with increased yields while maintaining low production and maintenance costs is highly important since plants are the major food source for humans and animals, as well as important producers of chemicals, pharmaceuticals, and fuels. Gene editing approaches, particularly the CRISPR-Cas system, are the preferred methods for improving crops, enabling quick, robust, and accurate gene manipulation. Nevertheless, new breeds of genetically modified crops have initiated substantial debates concerning their biosafety, commercial use, and regulation. Here, we discuss the challenges facing genetic engineering of crops by CRISPR-cas, and highlight the pros and cons of using this tool.

Extending the Host Range of Bacteriophage Particles for DNA Transduction.

A major limitation in using bacteriophage-based applications is their narrow host range. Approaches for extending the host range have focused primarily on lytic phages in hosts supporting their propagation rather than approaches for extending the ability of DNA transduction into phage-restrictive hosts. To extend the host range of T7 phage for DNA transduction, we have designed hybrid particles displaying various phage tail/tail fiber proteins. These modular particles were programmed to package and transduce DNA into hosts that restrict T7 phage propagation. We have also developed an innovative generalizable platform that considerably enhances DNA transfer into new hosts by artificially selecting tails that efficiently transduce DNA. In addition, we have demonstrated that the hybrid particles transduce desired DNA into desired hosts. This study thus critically extends and improves the ability of the particles to transduce DNA into novel phage-restrictive hosts, providing a platform for myriad applications that require this ability.

Repeat Size Determination by Two Molecular Rulers in the Type I-E CRISPR Array.

Prokaryotic adaptive immune systems are composed of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. These systems adapt to new threats by integrating short nucleic acids, termed spacers, into the CRISPR array. The functional motifs in the repeat and the mechanism by which a constant repeat size is maintained are still elusive. Here, through a series of mutations within the repeat of the CRISPR-Cas type I-E, we identify motifs that are crucial for adaptation and show that they serve as anchor sites for two molecular rulers determining the size of the new repeat. Adaptation products from various repeat mutants support a model in which two motifs in the repeat bind to two different sites in the adaptation complex that are 8 and 16 bp away from the active site. This model significantly extends our understanding of the adaptation process and broadens the scope of its applications.

Attenuated Weight Gain with the Novel Analog of Olanzapine Linked to Sarcosinyl Moiety (PGW5) Compared to Olanzapine.

Olanzapine-induced weight gain is associated with atherosclerosis, hypertension, dyslipidemia, and diabetes. We synthesized a novel antipsychotic drug (PGW5) possessing an olanzapine moiety linked to sarcosine, a glycine transporter 1 inhibitor. In this study, we compared the metabolic effects of PGW5 and olanzapine in a female rat model of weight gain. Female rats were treated daily with oral olanzapine (4 mg/kg), PGW5 (25 mg/kg), or vehicle for 16 days. Behavioral tests were conducted on days 12-14. Biochemical analyses were performed at the end of the treatment. A significant increase in body weight was observed in the olanzapine-treated group, while the PGW5 group did not differ from the controls. The open field test showed hypo-locomotion in the olanzapine-treated animals as compared to PGW5 and control groups. A significant increase in hypothalamic protein expression of the neuropeptide Y5 receptor and a decrease in pro-opiomelanocortin messenger ribonucleic acid (mRNA) levels were detected following PGW5 treatment, but not after olanzapine administration. PGW5 appears to possess minor metabolic effects compared with the parent compound olanzapine. The differential modulation of brain peptides associated with appetite regulation is possibly involved in the attenuation of metabolic effects by PGW5.