ArticleProceedings of the National Academy of Sciences of the United States of America2016
Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 27 papers.
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Who cites it
27 citing papers in PubMed, 65 citations in OpenAlex.
- Machine learning reveals sequence and methylation determinants of SaCas9-PAM interactions in bacteria.Nucleic acids research · 2026Article
- Modular determinants of cleavage preference in GIY-YIG nucleases revealed by block-based DNA shuffling and directed evolution.Nucleic acids research · 2026Article
- A buffer-tuning strategy to profile domain-specific activity of chimeric I-TevI/CRISPR gene editors in vitro.Scientific reports · 2025Article
- Advancing Precision Medicine: Recent Innovations in Gene Editing Technologies.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2025Review
- Multiplex CRISPR-Cas Genome Editing: Next-Generation Microbial Strain Engineering.Journal of agricultural and food chemistry · 2024Review
- A Photoresponsive Homing Endonuclease for Programmed DNA Cleavage.ACS synthetic biology · 2024Article
- Article
- A generalizable Cas9/sgRNA prediction model using machine transfer learning with small high-quality datasets.Nature communications · 2023Article
- Noncanonical functions of Ku may underlie essentiality in human cells.Scientific reports · 2023Article
- Metabolically-targeted dCas9 expression in bacteria.Nucleic acids research · 2023Article
- CRISPR/Cas9 System: A Potential Tool for Genetic Improvement in Floricultural Crops.Molecular biotechnology · 2022Review
- Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing.Genome biology · 2022Article
- Precise plant genome editing using base editors and prime editors.Nature plants · 2021Review
- Precise, predictable multi-nucleotide deletions in rice and wheat using APOBEC-Cas9.Nature biotechnology · 2020Article
- A simple and highly efficient method for multi-allelic CRISPR-Cas9 editing in primary cell cultures.Cancer reports (Hoboken, N.J.) · 2020Article
- Plasmid-based complementation of large deletions in Phaeodactylum tricornutum biosynthetic genes generated by Cas9 editing.Scientific reports · 2020Article
- Can Designer Indels Be Tailored by Gene Editing?: Can Indels Be Customized?BioEssays : news and reviews in molecular, cellular and developmental biology · 2019Article
- Efficient inter-species conjugative transfer of a CRISPR nuclease for targeted bacterial killing.Nature communications · 2019Article
- Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair.Nucleic acids research · 2019Article
- Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing.Genome biology · 2018Article
Corrections and comments
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Authors and funding
9 authors at 1 institution in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33- to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.
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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.