Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs.

CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence ('guide sequence') and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2'-O-methyl-3'-phosphonoacetate, or 'MP') incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications.

Synthesis of Small-Molecule/DNA Hybrids through On-Bead Amide-Coupling Approach.

Small molecule/DNA hybrids (SMDHs) have been considered as nanoscale building blocks for engineering 2D and 3D supramolecular DNA assembly. Herein, we report an efficient on-bead amide-coupling approach to prepare SMDHs with multiple oligodeoxynucleotide (ODN) strands. Our method is high yielding under mild and user-friendly conditions with various organic substrates and homo- or mixed-sequenced ODNs. Metal catalysts and moisture- and air-free conditions are not required. The products can be easily analyzed by LC-MS with accurate mass resolution. We also explored nanometer-sized shape-persistent macrocycles as novel multitopic organic linkers to prepare SMDHs. SMDHs bearing up to six ODNs were successfully prepared through the coupling of arylenethynylene macrocycles with ODNs, which were used to mediate the assembly of gold nanoparticles.

Highly Active Multidentate Ligand-Based Alkyne Metathesis Catalysts.

Alkyne metathesis catalysts composed of molybdenum(VI) propylidyne and multidentate tris(2-hydroxylbenzyl)methane ligands have been developed, which exhibit excellent stability (remains active in solution for months at room temperature), high activity, and broad functional-group tolerance. The homodimerization and cyclooligomerization of monopropynyl or dipropynyl substrates, including challenging heterocycle substrates (e.g., pyridine), proceed efficiently at 40-55 °C in a closed system. The ligand structure and catalytic activity relationship has been investigated, which shows that the ortho groups of the multidentate phenol ligands are critical to the stability and activity of such a catalyst system.

Covalent assembly of heterosequenced macrocycles and molecular cages through orthogonal dynamic covalent chemistry (ODCC).

Shape-persistent heterosequenced 2-D macrocycles and 3-D molecular cages have been prepared in one pot from two or three different monomers, through orthogonal dynamic covalent chemistry using dynamic imine and olefin metathesis.

Highly efficient one-pot synthesis of hetero-sequenced shape-persistent macrocycles through orthogonal dynamic covalent chemistry (ODCC).

A series of hetero-sequenced shape-persistent macrocycles with different shape, size, and backbone (functional group) symmetry have been synthesized through one-pot orthogonal dynamic covalent chemistry (ODCC) in good to excellent yields from readily accessible starting materials.