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Updated: Jan 2, 2026
Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
Published on: February 26, 2019
Trio deep-sequencing does not reveal unexpected off-target and on-target mutations in Cas9-edited rhesus monkeys
Xin Luo1,2,3, Yaoxi He1,2,3, Chao Zhang4
1State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, 650223, Kunming, China.
Abstract:
CRISPR-Cas9 is a widely-used genome editing tool, but its off-target effect and on-target complex mutations remain a concern, especially in view of future clinical applications. Non-human primates (NHPs) share close genetic and physiological similarities with humans, making them an ideal preclinical model for developing Cas9-based therapies. However, to our knowledge no comprehensive in vivo off-target and on-target assessment has been conducted in NHPs. Here, we perform whole genome trio sequencing of Cas9-treated rhesus monkeys. We only find a small number of de novo mutations that can be explained by expected spontaneous mutations, and no unexpected off-target mutations (OTMs) were detected. Furthermore, the long-read sequencing data does not detect large structural variants in the target region.
Insights
CRISPR-Cas9 genome editing in non-human primates showed no unexpected off-target mutations. Whole genome sequencing confirmed safety, with only spontaneous mutations detected, supporting its preclinical use.
Area of Science:
- Genomics
- Molecular Biology
- Primate Research
Background:
- CRISPR-Cas9 is a powerful genome editing tool with potential clinical applications.
- Concerns exist regarding off-target effects and complex mutations associated with CRISPR-Cas9.
- Non-human primates (NHPs) are valuable preclinical models due to their genetic and physiological similarity to humans.
Purpose of the Study:
- To conduct a comprehensive in vivo assessment of CRISPR-Cas9's off-target and on-target effects in non-human primates.
- To evaluate the safety and accuracy of CRISPR-Cas9 genome editing in a relevant preclinical model.
Main Methods:
- Whole genome trio sequencing was performed on CRISPR-Cas9-treated rhesus monkeys.
- Long-read sequencing was utilized to detect potential large structural variants.
Main Results:
- A small number of de novo mutations were identified, consistent with expected spontaneous mutation rates.
- No unexpected off-target mutations (OTMs) were detected across the genome.
- Long-read sequencing data did not reveal large structural variants at the target sites.
Conclusions:
- CRISPR-Cas9 genome editing in rhesus monkeys appears safe with respect to off-target mutations.
- The findings support the use of CRISPR-Cas9 in NHPs as a preclinical model for gene therapy development.
- Comprehensive genomic assessment is crucial for validating gene editing technologies in vivo.
