mRNA Transport and Local Translation in Glia

Though mRNA transport and local translation are extensively studied in neurons, emerging evidence supports that these cellular processes are also abundant in non-neuronal glial cells. Here, we explore mechanisms of mRNA transport and local translation in oligodendrocytes, astrocytes, microglia, radial glia, and their functions in development, structure, and intercellular interactions

CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets

Sensitive detection of off-target effects is important for translating CRISPR-Cas9 nucleases into human therapeutics. In vitro biochemical methods for finding off-targets offer potential advantages of greater reproducibility and scalability while avoiding limitations associated with strategies that require the culture and manipulation of living cells. Here we describe CIRCLE-seq (Circularization for In vitro Reporting of CLeavage Effects by sequencing), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRISPR-Cas9 genome-wide off-target mutations. In contrast to previously described in vitro methods, we show that CIRCLE-seq can be practiced using widely accessible next-generation sequencing technology and does not require reference genome sequence. Importantly, CIRCLE-seq can be used to identify off-target mutations associated with cell-type-specific SNPs, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides the most accessible, rapid and comprehensive method for identifying genome-wide off-target mutations of CRISPR-Cas9 described to date

Replacing Uridine with 2‑Thiouridine Enhances the Rate and Fidelity of Nonenzymatic RNA Primer Extension

The nonenzymatic replication of RNA oligonucleotides is thought to have played a key role in the origin of life prior to the evolution of ribozyme-catalyzed RNA replication. Although the copying of oligo-C templates by 2-methylimidazole-activated G monomers can be quite efficient, the copying of mixed sequence templates, especially those containing A and U, is particularly slow and error-prone. The greater thermodynamic stability of the 2-thio-U­(s²U):A base pair, relative to the canonical U:A base pair, suggests that replacing U with s²U might enhance the rate and fidelity of the nonenzymatic copying of RNA templates. Here we report that this single atom substitution in the activated monomer improves both the kinetics and the fidelity of nonenzymatic primer extension on mixed-sequence RNA templates. In addition, the mean lengths of primer extension products obtained with s²U is greater than those obtained with U, augmenting the potential for nonenzymatic replication of heritable function-rich sequences. We suggest that noncanonical nucleotides such as s²U may have played a role during the infancy of the RNA world by facilitating the nonenzymatic replication of genomic RNA oligonucleotides