Related Experiment Video
Updated: Nov 26, 2025

08:23
CIRCLE-Seq for Interrogation of Off-Target Gene Editing
Published on: November 1, 2024
1.1K
Tools for experimental and computational analyses of off-target editing by programmable nucleases
X Robert Bao1,2, Yidan Pan3, Ciaran M Lee4
1ILISATech, Houston, TX, USA.
Nature Protocols
|December 8, 2020
Summary
Genome editing tools show promise but off-target effects are a concern. Combining in silico and experimental methods with next-generation sequencing (NGS) is recommended for accurate off-target analysis in therapeutic applications.
Area of Science:
- Genomics
- Molecular Biology
- Biotechnology
Background:
- Genome editing technologies, such as CRISPR-Cas9, are transformative for life sciences and medicine.
- Off-target editing by programmable nucleases is a significant safety concern, particularly for therapeutic uses.
Purpose of the Study:
- To review and compare computational (in silico) and experimental tools for identifying and analyzing genome editing off-target effects.
- To highlight limitations in current methods for detecting low-frequency off-target editing events.
Main Methods:
- Review of existing in silico and experimental tools for off-target analysis.
- Comparison of the predictive accuracy of various tools for off-target editing sites.
- Assessment of next-generation sequencing (NGS) as a gold standard assay.
Main Results:
- Recent advances show good concordance between in silico and experimental tools for high-frequency off-target sites.
- No single tool accurately predicts low-frequency off-target editing events, posing a challenge for therapeutics.
- Amplicon-based NGS demonstrates high accuracy in quantifying off-target effects.
Conclusions:
- A combined approach using at least one in silico and one experimental tool is recommended for identifying potential off-target sites.
- Amplicon-based NGS should be employed as the gold standard for validating off-target effects.
- Future research should focus on expanding datasets, refining experimental techniques, and improving computational algorithms for more sensitive off-target detection.
More Related Videos
Related Concept Videos
CRISPR/Cas9 Genome Editing
1.1K
The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
1.1K
Restriction Enzymes
34.7K
Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called restriction sites. Such palindromic DNA sequences or inverted repeats are commonly found in regions of functional significance, such as the origin of replication, gene operator sites, and regions containing transcription termination signals.
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...
34.7K
CRISPR and crRNAs
18.2K
Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
18.2K
CRISPR
55.4K
Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
55.4K
Conservative Site-specific Recombination and Phase Variation
6.4K
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
6.4K

