Journal Article

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

den Hoed, Marcel, Ameur, Adam, Chin, Chen-Shan, Emmanouilidou, Anastasia, Feuk, Lars, Gyllensten, Ulf, Wilbe, Maria, Gudmundsson, Sanna, Bondeson, Marie-Louise, Johansson, Josefin, Bunikis, Ignas, Höijer, Ida, Häggqvist, Susana

2020

Overview

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.

• Publisher: BioMed Central,Springer Nature B.V,BMC
• Subject: Animal Genetics and Genomics / Base Sequence / Bioinformatics / Biomedical and Life Sciences / Computational Biology - methods / Computer applications / computer software / CRISPR / CRISPR-Cas Systems / CRISPR-Cas9 / Deoxyribonucleic acid / digestion / DNA / DNA sequencing / Evolutionary Biology / Fibroblasts / Gene deletion / Gene Editing - methods / genes / Genetic Variation / Genome editing / Genomes / Genomics / gRNA / HEK293 Cells / human cell lines / Human Genetics / Humans / Insertion / Life Sciences / Long-read sequencing / Microbial Genetics and Genomics / Mutation / Nano-OTS / Nanopore Sequencing / Off-target / On-target / PacBio sequencing / Plant Genetics and Genomics / prediction / Ribonucleic acid / RNA / RNA, Guide, CRISPR-Cas Systems / Sequence Analysis, DNA / Single molecule sequencing / SMRT-OTS / Software