Simple and Rapid Assembly of TALE Modules Based on the Degeneracy of the Codons and Trimer Repeats

Transcription activator-like effectors (TALEs) have been effectively used for targeted genome editing, transcriptional regulation, epigenetic modification, and locus-specific DNA imaging. However, with the advent of the clustered regularly interspaced short palindromic repeat/Cas9 system, an easy-to-use tool with the same function as TALEs, TALEs have recently been abandoned because of their complexity, time consumption, and difficult handling in common labs. Here, we described a degenerated codon-based TALE assembly system for simple, rapid, and efficient TALE assembly. TALE trimers with nonrepetitive DNA sequences were amplified by PCR and sequentially assembled via Gibson assembly. Our method is cost-effective, requires only commonly used basic molecular biology reagents, and takes only 2 h from target sequence analysis to completion. This simple, rapid, and lab-friendly TALE assembly method will restore the value of TALEs in DNA targeting.

Cell cycle– and genomic distance–dependent dynamics of a discrete chromosomal region

In contrast to the well-studied condensation and folding of chromosomes during mitosis, their dynamics during interphase are less understood. We deployed a CRISPR-based DNA imaging system to track the dynamics of genomic loci situated kilobases to megabases apart on a single chromosome. Two distinct modes of dynamics were resolved: local movements as well as ones that might reflect translational movements of the entire domain within the nucleoplasmic space. The magnitude of both of these modes of movements increased from early to late G1, whereas the translational movements were reduced in early S phase. The local fluctuations decreased slightly in early S and more markedly in mid-late S. These newly observed movements and their cell cycle dependence suggest the existence of a hitherto unrecognized compaction–relaxation dynamic of the interphase chromosome fiber, operating concurrently with changes in the extent of overall movements of loci in the 4D genome.

A dinuclear ruthenium(ii) phototherapeutic that targets duplex and quadruplex DNA

A previously reported dinuclear DNA imaging probe has been converted into a phototherapeutic through the incorporation of RuII(TAP)2 fragments (TAP = 1,4,5,8-3 tetraazaphenanthrene).

CRISPR-Based DNA Imaging in Living Cells Reveals Cell Cycle-Dependent Chromosome Dynamics

SUMMARYIn contrast to the well-studied condensation and folding of chromosomes during mitosis, their dynamics in interphase are less understood. We developed a sensitive, multicolor system, CRISPR-Sirius, allowing the real-time tracking of the dynamics of chromosomal loci. We tracked loci kilobases to megabases apart and found significant variation in the inter-locus distances of each pair, indicating differing degrees of DNA contortion. We resolved two distinct modes of dynamics of loci: saltatory local movements as well as translational movements of the domain. The magnitude of both of these modes of movements increased from early to late G1, whereas the translational movements were reduced in early S. The local fluctuations decreased slightly in early S and more markedly in mid-late S. These newly observed movements and their cell cycle-dependence are indicative of a hitherto unrecognized compaction-relaxation dynamic of the chromosomal fiber operating concurrently with changes in the extent of observed genomic domain movements.IN BRIEFDistinct chromosome folding and dynamics during cell cycle progression were dissected by CRISPR-Sirius DNA imaging in living cells.HIGHLIGHTSCRISPR-Sirius allows tracking of pairs of chromosomal loci having kilobase to megabase inter-locus distancesPair-wise tracking of loci allows measurement of both local and domain dynamicsChromosomal fiber relaxation is positively correlated with local dynamicsGenomic region size contributes to local and domain movementsDistinct chromosome dynamics were uncovered during cell cycle progression in interphase

Polymer conformation during flow in porous media

Molecular conformations of individual polymers during flow through porous media are directly observed by single-DNA imaging in microfluidics.