ArticleNucleic acids research2020
5' modifications to CRISPR-Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage.
Article in Nucleic acids research, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 28 papers.
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Who cites it
28 citing papers in PubMed, 50 citations in OpenAlex.
- CRISPR activation reveals SOX5/6/9 as key transcriptional regulators directing iPSC-derived cells toward a notochordal lineage.Stem cell reports · 2026Article
- Cellular-state control using ribozyme-scaffolded miRNA-sensing and CRISPR-mediated actuation.Cell reports methods · 2026Article
- CRISPR/Cas9 Genome Editing in Oncology: Mechanisms, Therapeutic Platforms and Translational Challenges.Molecular biotechnology · 2026Review
- CRISPR-Cas9 trans-cleavage is hindered by a flanked R-loop, an elongated spacer, and an inactive HNH domain.Nature communications · 2026Article
- Research progress and applications of gene activation editing technology in crops.Frontiers in plant science · 2026Review
- CRISPR-Cas12a REC2-Nuc interactions drive target-strand cleavage and constrain trans cleavage.Nucleic acids research · 2025Article
- Article
- Engineering circular guide RNA and CRISPR-Cas13d-encoding mRNA for the RNA editing ofbioRxiv : the preprint server for biology · 2025Article
- Massively parallel in vivo Perturb-seq screening.Nature protocols · 2025Review
- Single-molecule perspectives of CRISPR/Cas systems: target search, recognition, and cleavage.BMB reports · 2025Review
- Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo.Nature biotechnology · 2024Article
- Re-engineered guide RNA enables DNA loops and contacts modulating repression in E. coli.Nucleic acids research · 2024Article
- Compartmentalized CRISPR Reactions (CCR) for High-Throughput Screening of Guide RNA Potency and Specificity.bioRxiv : the preprint server for biology · 2024Article
- Review
- Delivery of gene editing therapeutics.Nanomedicine : nanotechnology, biology, and medicine · 2023Review
- Vector enabled CRISPR gene editing - A revolutionary strategy for targeting the diversity of brain pathologies.Coordination chemistry reviews · 2023Article
- Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs.Nature biomedical engineering · 2023Article
- Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria.Nature communications · 2023Article
- Single Molecule FRET Analysis of CRISPR Cas9 Single Guide RNA Folding Dynamics.The journal of physical chemistry. B · 2023Article
- Building Blocks of Artificial CRISPR-Based Systems beyond Nucleases.International journal of molecular sciences · 2022Review
Corrections and comments
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Authors and funding
8 authors at 2 institutions in 2 countries.
Funding
Abstract
A key aim in exploiting CRISPR-Cas is gRNA engineering to introduce additional functionalities, ranging from individual nucleotide changes that increase efficiency of on-target binding to the inclusion of larger functional RNA aptamers or ribonucleoproteins (RNPs). Cas9-gRNA interactions are crucial for complex assembly, but several distinct regions of the gRNA are amenable to modification. We used in vitro ensemble and single-molecule assays to assess the impact of gRNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that RNP formation was unaffected by any of our modifications. R-loop formation and DNA cleavage activity were also essentially unaffected by modification of the Upper Stem, first Hairpin and 3' end. In contrast, we found that 5' additions of only two or three nucleotides could reduce R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5' end of a gRNA still supported RNP formation but produced a stable ∼9 bp R-loop that could not activate DNA cleavage. Consideration of these observations will assist in successful gRNA design.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.