Ishita Jain

The Escherichia coli type I-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interference in vivo Shortened crRNAs assemble into altered-stoichiometry Cascade effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multisubunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.

Type VI CRISPR-Cas systems are among the few CRISPR varieties that target exclusively RNA. The CRISPR RNA–guided, sequence-specific binding of target RNAs, such as phage transcripts, activates the type VI effector, Cas13. Once activated, Cas13 causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from the phage spread. We show here that the principal form of collateral RNA degradation elicited by Leptotrichia shahii Cas13a expressed in Escherichia coli cells is the cleavage of anticodons in a subset of transfer RNAs (tRNAs) with uridine-rich anticodons. This tRNA cleavage is accompanied by inhibition of protein synthesis, thus providing defense from the phages. In addition, Cas13a-mediated tRNA cleavage indirectly activates the RNases of bacterial toxin-antitoxin modules cleaving messenger RNA, which could provide a backup defense. The mechanism of Cas13a-induced antiphage defense resembles that of bacterial anticodon nucleases, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module encompassing an anticodon nuclease.

Target binding by CRISPR-Cas ribonucleoprotein effectors is initiated by the recognition of double-stranded PAM motifs by the Cas protein moiety followed by destabilization, localized melting, and interrogation of the target by the guide part of CRISPR RNA moiety. The latter process depends on seed sequences, parts of the target that must be strictly complementary to CRISPR RNA guide. Mismatches between the target and CRISPR RNA guide outside the seed have minor effects on target binding, thus contributing to off-target activity of CRISPR-Cas effectors. Here, we define the seed sequence of the Type V Cas12b effector from Bacillus thermoamylovorans. While the Cas12b seed is just five bases long, in contrast to all other effectors characterized to date, the nucleotide base at the site of target cleavage makes a very strong contribution to target binding. The generality of this additional requirement was confirmed during analysis of target recognition by Cas12b effector from Alicyclobacillus acidoterrestris. Thus, while the short seed may contribute to Cas12b promiscuity, the additional specificity determinant at the site of cleavage may have a compensatory effect making Cas12b suitable for specialized genome editing applications.

Skeletal muscle regeneration remains a clinical unmet need for volumetric muscle loss and atrophy where muscle function cannot be restored to prior capacity. Current experimental approaches do not account for the complex microenvironmental factors that modulate myogenesis. In this study we developed a biomimetic tissue chip platform to systematically study the combined effects of the extracellular matrix (ECM) microenvironment and mechanical strain on myogenesis of murine myoblasts. Using stretchable tissue chips composed of collagen I (C), fibronectin (F) and laminin (L), as well as their combinations thereof, we tested the addition of mechanical strain regimens on myogenesis at the transcriptomic and translational levels. Our results show that ECMs have a significant effect on myotube formation in C2C12 murine myoblasts. Under static conditions, laminin substrates induced the longest myotubes, whereas fibronectin produced the widest myotubes. Combinatorial ECMs showed non-intuitive effects on myotube formation. Genome-wide analysis revealed the upregulation in actin cytoskeletal related genes that are suggestive of myogenesis. When mechanical strain was introduced to C + F + L combinatorial ECM substrates in the form of constant or intermittent uniaxial strain at low (5%) and high (15%) levels, we observed synergistic enhancements in myotube width, along with transcriptomic upregulation in myosin heavy chain genes. Together, these studies highlight the complex role of microenvironmental factors such as ECM interactions and strain on myotube formation and the underlying signaling pathways.

In space, astronauts are exposed to a large doses of ionizing radiation which can cause various health problems. The drug delivery of antioxidants and anti-inflammatory substances is a promising countermeasure for the harmful cellular effects of radiation exposure. Curcumin is a polyphenol derived from the rhizome of the turmeric plant that has strong antioxidant capabilities. The therapeutic potential of this radical scavenging drug is limited by poor uptake in the body due to its insolubility in water, rapid metabolism by the intestinal mucosa and liver, and quick excretion. Drug delivery vehicles can be used to enhance the bioavailability of curcumin. Albumin, a biodegradable and non-immunogenic plasma protein, can be utilized as a drug delivery vehicle. Curcumin binds to albumin’s hydrophobic pockets, which increases its solubility and decreases its rate of degradation in physiological conditions. Curcumin was solubilized with 0.5% (w/v) fatty acid free human serum albumin (FAF HSA) and spray dried to form a dry powder of particles. To produce particles with a smooth and spherical morphology, 0.05% (v/v) Tween® 20 was included in the solution. Curcumin release from these particles followed a first-order release profile (Ct/Cinf = 1 - ekt, t = time in min) with k = 0.065 ± 0.003. To alter the release of curcumin from the spray dried particles, 0.5% FAF HSA was crosslinked using 0.01M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 5 mM Nhydroxysulfosuccinimide. This crosslinking method was confirmed by native polyacrylamide gel electrophoresis (PAGE) and differential scanning calorimetry (DSC). For the native PAGE results, multiple protein bands at high molecular weights were observed for the crosslinked FAF HSA and a single protein band for the uncrosslinked FAF HSA. This suggested that multiple FAF HSA molecules had been crosslinked to each other. DSC results reported a significant increase in melting point from 53.60 ± 1.35 °C to 63.82 ± 2.34 °C of spray dried FAF HSA particles, further confirming the crosslinking method. Curcumin was bound to the crosslinked FAF HSA in the presence of 0.05% Tween® 20 and spray dried. The resulting particles showed a less uniform morphology and curcumin release followed a first-order profile with a significantly lower k = 0.049 ± 0.004. Future work to improve the morphology of the crosslinked FAF HSA particles and increase the level of crosslinking to further slow curcumin release will enhance the applicability of these particles in mitigating radiation-induced cell damage.