{"entity": "publication", "iuid": "debf73d8bffa429fb06e5d42eb9d808d", "timestamp": "2026-09-30T03:00:45.039Z", "links": {"self": {"href": "https://publications-affiliated.scilifelab.se/publication/debf73d8bffa429fb06e5d42eb9d808d.json"}, "display": {"href": "https://publications-affiliated.scilifelab.se/publication/debf73d8bffa429fb06e5d42eb9d808d"}}, "title": "Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity.", "authors": [{"family": "H\u00f6ijer", "given": "Ida", "initials": "I", "orcid": "0000-0002-3915-3384", "researcher": {"href": "https://publications-affiliated.scilifelab.se/researcher/fa1a8d7500f147cb852a2e9c3e8617c5.json"}}, {"family": "Johansson", "given": "Josefin", "initials": "J"}, {"family": "Gudmundsson", "given": "Sanna", "initials": "S"}, {"family": "Chin", "given": "Chen-Shan", "initials": "CS"}, {"family": "Bunikis", "given": "Ignas", "initials": "I"}, {"family": "H\u00e4ggqvist", "given": "Susana", "initials": "S"}, {"family": "Emmanouilidou", "given": "Anastasia", "initials": "A"}, {"family": "Wilbe", "given": "Maria", "initials": "M"}, {"family": "den Hoed", "given": "Marcel", "initials": "M"}, {"family": "Bondeson", "given": "Marie-Louise", "initials": "ML"}, {"family": "Feuk", "given": "Lars", "initials": "L"}, {"family": "Gyllensten", "given": "Ulf", "initials": "U"}, {"family": "Ameur", "given": "Adam", "initials": "A"}], "type": "journal article", "published": "2020-12-01", "journal": {"title": "Genome Biol.", "issn": "1474-760X", "volume": "21", "issue": "1", "pages": "290", "issn-l": "1474-7596"}, "abstract": "One 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.\n\nThe 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.\n\nAmplification-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.", "doi": "10.1186/s13059-020-02206-w", "pmid": "33261648", "labels": [], "xrefs": [{"db": "pmc", "key": "PMC7706270"}, {"db": "pii", "key": "10.1186/s13059-020-02206-w"}], "notes": [], "created": "2026-09-23T08:35:11.688Z", "modified": "2026-09-23T08:35:11.747Z"}