Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity
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RESEARCH
Open Access
Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity Ida Höijer1* , Josefin Johansson1, Sanna Gudmundsson1,2,3, Chen-Shan Chin4, Ignas Bunikis1, Susana Häggqvist1, Anastasia Emmanouilidou1,5, Maria Wilbe1, Marcel den Hoed1,5, Marie-Louise Bondeson1, Lars Feuk1, Ulf Gyllensten1 and Adam Ameur1,6* * Correspondence: ida.hoijer@igp. uu.se; [email protected] 1 Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Full list of author information is available at the end of the article
Abstract 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. Keywords: CRISPR-Cas9, On-target, Off-target, Long-read sequencing, Single molecule sequencing, PacBio sequencing, Nanopore sequencing, SMRT-OTS, Nano-OTS
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