ArticleGenome biology2020
Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity.
Article in Genome biology, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 40 papers, 1 of them a synthesis that pooled it.
What it found
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
The trial behind it
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Who cites it
40 citing papers in PubMed, 1 synthesis or guideline pooled it, 66 citations in OpenAlex.
- Pooled it
- Chemical and Structural Engineering of Guide RNAs for Precision Genome Editing: From Design Principles to Clinical Applications.Chemical biology & drug design · 2026Review
- Article
- RiSpy: a feature selection-based fingerprinting framework for accurate identification of genome-edited rice lines.Briefings in bioinformatics · 2026Article
- Multilevel characterization of genome editor nuclease activity with BreakTag.Nature protocols · 2026Review
- MASTR-seq enables multiplexed analysis of short tandem repeats with sequencing.Cell reports methods · 2026Article
- Efficient multi-kilobase knock-ins in mice and cell lines using CRISPR/Cas9 and rAAV donors with unbiased whole-genome characterization by LOCK-seq.Nucleic acids research · 2026Article
- Enhancing CRISPR Homology Directed Repair in IAL-PiD2 Insect Cells via Reagent Delivery Optimization and Cell Synchronization.Biochemical engineering journal · 2026Article
- De-extinction: how reviving the past is revolutionizing the future of conservation biology.The Journal of reproduction and development · 2026Review
- Revealing genetic drivers of ovarian cancer and chemoresistance: insights from whole-genome CRISPR-knockout library screens.Cellular oncology (Dordrecht, Netherlands) · 2025Review
- Off-target effects in CRISPR-Cas genome editing for human therapeutics: Progress and challenges.Molecular therapy. Nucleic acids · 2025Review
- Precise measurement of CRISPR genome editing outcomes through single-cell DNA sequencing.Molecular therapy. Methods & clinical development · 2025Article
- A photo-inducible nano-switch for spatiotemporal controlled gene editing activation.Materials today. Bio · 2025Article
- Suitability of Real-Time PCR Methods for New Genomic Technique Detection in the Context of the European Regulations: A Case Study in Arabidopsis.International journal of molecular sciences · 2025Article
- A bioinformatic analysis of gene editing off-target loci altered by common polymorphisms, using 'PopOff'.Journal of the Royal Society of New Zealand · 2025Article
- Predicting CRISPR-Cas9 off-target effects in human primary cells using bidirectional LSTM with BERT embedding.Bioinformatics advances · 2025Article
- Advances in nanomaterial-mediated CRISPR/Cas delivery: from lipid nanoparticles to vesicle-derived systems.Frontiers in bioengineering and biotechnology · 2025Review
- Combining Off-flow, a Nextflow-coded program, and whole genome sequencing reveals unintended genetic variation in CRISPR/Cas-edited iPSCs.Computational and structural biotechnology journal · 2024Article
- Applications of CRISPR Technologies in Forestry and Molecular Wood Biotechnology.International journal of molecular sciences · 2024Review
- Enhancing genome editing in hPSCs through dual inhibition of DNA damage response and repair pathways.Nature communications · 2024Article
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
13 authors at 3 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundOne ongoing concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target activity is challenging. Here, we present SMRT-OTS and Nano-OTS, two novel, amplification-free, long-read sequencing protocols for detection of gRNA-driven digestion of genomic DNA by Cas9 in vitro.
resultsThe methods are assessed using the human cell line HEK293, re-sequenced at 18x coverage using highly accurate HiFi SMRT reads. SMRT-OTS and Nano-OTS are first applied to three different gRNAs targeting HEK293 genomic DNA, resulting in a set of 55 high-confidence gRNA cleavage sites identified by both methods. Twenty-five of these sites are not reported by off-target prediction software, either because they contain four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. Additional experiments reveal that 85% of Cas9 cleavage sites are also found by other in vitro-based methods and that on- and off-target sites are detectable in gene bodies where short-reads fail to uniquely align. Even though SMRT-OTS and Nano-OTS identify several sites with previously validated off-target editing activity in cells, our own CRISPR-Cas9 editing experiments in human fibroblasts do not give rise to detectable off-target mutations at the in vitro-predicted sites. However, indel and structural variation events are enriched at the on-target sites.
conclusionsAmplification-free long-read sequencing reveals Cas9 cleavage sites in vitro that would have been difficult to predict using computational tools, including in dark genomic regions inaccessible by short-read sequencing.
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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.