CRISPR (clustered regularly interspaced short palindromic repeat) endonucleases are at the forefront of biotechnology, synthetic biology and gene editing. Methods for controlling enzyme properties promise to improve existing applications and enable new technologies. CRISPR enzymes rely on RNA cofactors to guide catalysis. Therefore, chemical modification of the guide RNA can be used to characterize structure-activity relationships within CRISPR ribonucleoprotein (RNP) enzymes and identify compatible chemistries for controlling activity. Here, we introduce chemical modifications to the sugar–phosphate backbone of Streptococcus pyogenes Cas9 CRISPR RNA (crRNA) to probe chemical and structural requirements. Ribose sugars that promoted or accommodated A-form helical architecture in and around the crRNA ‘seed’ region were tolerated best. A wider range of modifications were acceptable outside of the seed, especially D-2′-deoxyribose, and we exploited this property to facilitate exploration of greater chemical diversity within the seed. 2′-fluoro was the most compatible modification whereas bulkier O-methyl sugar modifications were less tolerated. Activity trends could be rationalized for selected crRNAs using RNP stability and DNA target binding experiments. Cas9 activity in vitro tolerated most chemical modifications at predicted 2′-hydroxyl contact positions, whereas editing activity in cells was much less tolerant. The biochemical principles of chemical modification identified here will guide CRISPR-Cas9 engineering and enable new or improved applications.

Friedreich's Ataxia (FA) is an inherited neurologic disorder caused by an expanded GAA repeat within intron 1 of the frataxin (FXN) gene that reduces expression of FXN protein. Agents that increase expression of FXN have the potential to alleviate the disease. We previously reported that duplex RNAs (dsRNAs) and antisense oligonucleotides (ASOs) complementary to the GAA repeat could enhance expression of FXN protein. We now explore the potential of a diverse group of chemically modified dsRNAs and ASOs to define the breadth of repeat-targeted synthetic nucleic acids as a platform for therapeutic development for FA. ASOs and dsRNAs can activate FXN protein expression in FA patient-derived cell lines that possess varied numbers of GAA repeats. Increased FXN protein expression was achieved by ASOs incorporating diverse chemical modifications with low nanomolar potencies, suggesting substantial flexibility in choosing compounds for further chemical optimization and animal studies. Our data encourage further development of ASOs as agents to treat FA.

We report the general and rapid synthesis of short DNA fragments of controllable sequence and length using multi-step, one-pot mechanochemical reactions, without bulk solvent or the need to isolate intermediates. We demonstrate, for the first time, the multi-step ball milling synthesis of DNA dimers and trimers via phosphoramidite and H-phosphonate chemistries. The use of mechanochemistry allowed for coupling of phosphoramidite monomers to the 5'-hydroxyl group of nucleosides, iodine/water oxidation of the resulting phosphite triester linkage, and removal of the 5'-dimethoxy (DMTr) protecting group in situ and in good yields (up to 60% over three steps) to produce DNA dimers in one-pot manner. Sulphurization of phosphite triester linkages was possible using elemental sulfur yielding the corresponding phosphorothioate DNA dimers in good yield (up to 80% over two steps). By using H-phosphonate chemistry under milling conditions, it was possible to couple, protect the H-phosphonate linkage, and remove the 5'-DMTr protecting group in situ, enabling an one-pot process with good yields comparable to existing solvent-based procedures (up to 65% over three steps, or ca. 87% per step). This work opens the door to creation of solvent-free methodologies for the assembly of complex DNA and RNA therapeutics. File list (2) download file view on ChemRxiv ChemRxiv_manuscript.pdf (537.19 KiB) download file view on ChemRxiv SI_ChemRxiv.pdf (1.50 MiB)

Clustered regularly interspaced short palindromic repeat (CRISPR) RNAs and their associated effector (Cas) enzymes are being developed into promising therapeutics to treat disease. However, CRISPR-Cas enzymes might produce unwanted gene editing or dangerous side effects. Drug-like molecules that can inactivate CRISPR-Cas enzymes could help facilitate safer therapeutic development. Based on the requirement of guide RNA and target DNA interaction by Cas enzymes, we rationally designed small nucleic acid-based inhibitors (SNuBs) of Streptococcus pyogenes (Sp) Cas9. Inhibitors were initially designed as 2¢-O-methyl-modified oligonucleotides that bound the CRISPR RNA guide sequence (anti-guide) or repeat sequence (anti-tracr), or DNA oligonucleotides that bound the protospacer adjacent motif (PAM)-interaction domain (anti-PAM) of SpCas9. Coupling anti-PAM and anti-tracr modules together was synergistic and resulted in high binding affinity and efficient inhibition of Cas9 DNA cleavage activity. Incorporating 2¢F-RNA and locked nucleic acid nucleotides into the anti-tracr module resulted in greater inhibition as well as dose-dependent suppression of gene editing in human cells. CRISPR SNuBs provide a platform for rational design of CRISPR-Cas enzyme inhibitors that should translate to other CRISPR effector enzymes and enable better control over CRISPR-based applications.

The continuous evolution of SARS-CoV-2 variants complicates efforts to combat the ongoing pandemic, underscoring the need for a dynamic platform for the rapid development of pan-viral variant therapeutics. Oligonucleotide therapeutics are enhancing the treatment of numerous diseases with unprecedented potency, duration of effect, and safety. Through the systematic screening of hundreds of oligonucleotide sequences, we identified fully chemically stabilized siRNAs and ASOs that target regions of the SARS-CoV-2 genome conserved in all variants of concern, including delta and omicron. We successively evaluated candidates in cellular reporter assays, followed by viral inhibition in cell culture, with eventual testing of leads for in vivo antiviral activity in the lung. Previous attempts to deliver therapeutic oligonucleotides to the lung have met with only modest success. Here, we report the development of a platform for identifying and generating potent, chemically modified multimeric siRNAs bioavailable in the lung after local intranasal and intratracheal delivery. The optimized divalent siRNAs showed robust antiviral activity in human cells and mouse models of SARS-CoV-2 infection and represent a new paradigm for antiviral therapeutic development for current and future pandemics.