ArticleNucleic acids research2019
Extensive CRISPR RNA modification reveals chemical compatibility and structure-activity relationships for Cas9 biochemical activity.
Article in Nucleic acids research, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 36 papers.
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Who cites it
36 citing papers in PubMed, 64 citations in OpenAlex.
- Emerging strategies and innovations in circular RNA synthesis.Molecular therapy. Nucleic acids · 2026Review
- CRISPR RNA architecture steers Cas9 catalysis and fidelity via a guide repeat clasp motif.Nucleic acids research · 2026Article
- Fully Modified SpyCas9 Guide RNAs Enable Robust Genome Editing In Cells and In Vivo.bioRxiv : the preprint server for biology · 2026Article
- CRISPR-AuNP: physicochemical optimization of a gold nanoparticle platform for cost-effective and modular non-viral gene editing in HSPCs.Gene therapy · 2026Article
- Chemical control of 2'-hydroxyl-dependent Cas9 target engagement enables CRISPR RNA ribose replacement.bioRxiv : the preprint server for biology · 2026Article
- Synthesis of long and functionally active RNAs facilitated by acetal levulinic ester chemistry.Nucleic acids research · 2026Article
- Bio-orthogonal chemistry-based strategy to Turn-OFF CRISPR-Cas9 activity in solution and live cells.NAR molecular medicine · 2026Article
- A guide RNA repeat checkpoint steers CRISPR-Cas9 catalysis.bioRxiv : the preprint server for biology · 2025Article
- Coumarin-Based Photolabile Solid Support Facilitates Nonchromatographic Purification of RNA Oligonucleotides.The Journal of organic chemistry · 2025Article
- CRISPR-Cas Systems: A Functional Perspective and Innovations.International journal of molecular sciences · 2025Review
- Chemical engineering of CRISPR-Cas systems for therapeutic application.Nature reviews. Drug discovery · 2025Review
- Cas9 RNP Physiochemical Analysis for Enhanced CRISPR-AuNP Assembly and Function.bioRxiv : the preprint server for biology · 2024Article
- Recent advances in nanoparticulate RNA delivery systems.Proceedings of the National Academy of Sciences of the United States of America · 2024Article
- Emerging Gene Therapeutics for Epidermolysis Bullosa under Development.International journal of molecular sciences · 2024Review
- Self-delivering, chemically modified CRISPR RNAs for AAV co-delivery and genome editing in vivo.Nucleic acids research · 2024Article
- Harnessing non-Watson-Crick's base pairing to enhance CRISPR effectors cleavage activities and enable gene editing in mammalian cells.Proceedings of the National Academy of Sciences of the United States of America · 2024Article
- A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing inFrontiers in plant science · 2023Article
- Bio-Orthogonal Chemistry Conjugation Strategy Facilitates Investigation of N-methyladenosine and Thiouridine Guide RNA Modifications on CRISPR Activity.The CRISPR journal · 2022Article
- Improving Stability and Specificity of CRISPR/Cas9 System by Selective Modification of Guide RNAs with 2'-fluoro and Locked Nucleic Acid Nucleotides.International journal of molecular sciences · 2022Article
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Corrections and comments
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Authors and funding
12 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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.
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