Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

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Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

Höijer, Ida (author): Uppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik

Johansson, Josefin (author): Uppsala universitet,Medicinsk genetik och genomik,Science for Life Laboratory, SciLifeLab

Gudmundsson, Sanna, 1989- (author): Uppsala universitet,Medicinsk genetik och genomik,Science for Life Laboratory, SciLifeLab,Program in Medical and Population Genetics, Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA; Division of Genetics and Genomics, Boston Children’s Hospital, Boston, MA, USA

Chin, Chen-Shan (author)

Bunikis, Ignas (author): Uppsala universitet,Science for Life Laboratory, SciLifeLab,Institutionen för immunologi, genetik och patologi

Häggqvist, Susana (author): Uppsala universitet,Institutionen för immunologi, genetik och patologi,Science for Life Laboratory, SciLifeLab

Emmanouilidou, Anastasia (author): Uppsala universitet,Medicinsk genetik och genomik,Science for Life Laboratory, SciLifeLab

Wilbe, Maria (author): Uppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik

den Hoed, Marcel, 1980- (author): Uppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik

Bondeson, Marie-Louise, 1960- (author): Uppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik

Feuk, Lars (author): Uppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik

Gyllensten, Ulf (author): Uppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik

Ameur, Adam (author): Uppsala universitet,Institutionen för immunologi, genetik och patologi,Science for Life Laboratory, SciLifeLab,Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Australia

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2020-12-01
2020
English.
In: Genome Biology. - : Springer Science and Business Media LLC. - 1465-6906 .- 1474-760X. ; 21:1

Related links:: https://doi.org/10.1...; show more...; https://uu.diva-port... (primary) (Raw object); https://genomebiolog...; https://urn.kb.se/re...; https://doi.org/10.1...; show less...

Journal article (peer-reviewed)

Abstract Subject headings

BACKGROUND: 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.RESULTS: The 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.CONCLUSIONS: Amplification-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.

Subject headings

MEDICIN OCH HÄLSOVETENSKAP -- Medicinska och farmaceutiska grundvetenskaper -- Cell- och molekylärbiologi (hsv//swe)
MEDICAL AND HEALTH SCIENCES -- Basic Medicine -- Cell and Molecular Biology (hsv//eng)
MEDICIN OCH HÄLSOVETENSKAP -- Medicinska och farmaceutiska grundvetenskaper -- Medicinsk genetik (hsv//swe)
MEDICAL AND HEALTH SCIENCES -- Basic Medicine -- Medical Genetics (hsv//eng)

Keyword

CRISPR-Cas9
Long-read sequencing
Nano-OTS
Nanopore sequencing
Off-target
On-target
PacBio sequencing
SMRT-OTS
Single molecule sequencing

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By the author/editor: Höijer, Ida; Johansson, Josef ...; Gudmundsson, San ...; Chin, Chen-Shan; Bunikis, Ignas; Häggqvist, Susan ...; show more...; Emmanouilidou, A ...; Wilbe, Maria; den Hoed, Marcel ...; Bondeson, Marie- ...; Feuk, Lars; Gyllensten, Ulf; Ameur, Adam; show less...

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MEDICAL AND HEALTH SCIENCES: MEDICAL AND HEAL ...; and Basic Medicine; and Cell and Molecul ...

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