CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets
| Autor: | Shengdar Q. Tsai, Jose Malagon-Lopez, Martin J. Aryee, Ved V Topkar, Nhu T. Nguyen, J. Keith Joung |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Sequence analysis Sensitivity and Specificity Biochemistry Genome Article Off targets 03 medical and health sciences CRISPR Clustered Regularly Interspaced Short Palindromic Repeats Molecular Biology Genetics Nuclease Deoxyribonucleases biology Chromosome Mapping High-Throughput Nucleotide Sequencing Reproducibility of Results Sequence Analysis DNA Cell Biology In vitro Highly sensitive 030104 developmental biology biology.protein CRISPR-Cas Systems Biotechnology Reference genome |
| Zdroj: | Nature methods |
| ISSN: | 1548-7105 1548-7091 |
| Popis: | Sensitive detection of off-target effects is important for translating CRISPR-Cas9 nucleases into human therapeutics. In vitro biochemical methods for finding off-targets offer the potential advantages of greater reproducibility and scalability while avoiding limitations associated with strategies that require the culture and manipulation of living cells. Here we describe circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRISPR-Cas9 genome-wide off-target mutations. In contrast to previously described in vitro methods, we show that CIRCLE-seq can be practiced using widely accessible next-generation sequencing technology and does not require reference genome sequences. Importantly, CIRCLE-seq can be used to identify off-target mutations associated with cell-type-specific single-nucleotide polymorphisms, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides an accessible, rapid, and comprehensive method for identifying genome-wide off-target mutations of CRISPR-Cas9. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full text from SpringerLink