| Title |
Off-target detection of CRISPR-Cas9 nuclease in vitro with CROFT-Seq |
| Authors |
Toliušis, Paulius ; Grybauskas, Algirdas ; Šinkūnas, Tomas ; Karvelis, Tautvydas ; Sasnauskas, Giedrius ; Zaremba, Mindaugas |
| DOI |
10.1093/synbio/ysag006 |
| Full Text |
|
| Is Part of |
Synthetic biology.. Oxford : Oxford University Press. 2026, vol. 11, iss. 1, art. no. ysag006, p. 1-11.. eISSN 2397-7000 |
| Keywords [eng] |
Cas9 ; in vitro ; off-targets ; CROFT-Seq |
| Abstract [eng] |
Programmable CRISPR-Cas9 nucleases have become invaluable tools for genome editing. However, off-target cleavage by these nucleases can lead to unintended changes in the edited genome. Detection of off-target sites is critical to make genome editing technology safe and predictable. Although current in vitro methods for off-target detection can identify these sites, they are time-consuming and complex. Here, we present CROFT-Seq (CRISPR nuclease off-target detection by sequencing), a sensitive, rapid, and affordable assay for the genome-wide detection of Cas9 off-target sites in vitro. CROFT-Seq performs comparably to the commonly used in vitro methods and serves as a valuable and efficient tool for the rapid assessment of genome-editing nuclease specificity. Notably, a high proportion of the top-ranked off-target sites identified by CROFT-Seq were validated in cells, highlighting its strong predictive performance. |
| Published |
Oxford : Oxford University Press |
| Type |
Journal article |
| Language |
English |
| Publication date |
2026 |
| CC license |
|
