PT  - JOURNAL ARTICLE
AU  - Geng, Keyi
AU  - Merino, Lara G.
AU  - Wedemann, Linda
AU  - Martens, Aniek
AU  - Sobota, Małgorzata
AU  - Sanchez, Yerma P.
AU  - Søndergaard, Jonas Nørskov
AU  - White, Robert J.
AU  - Kutter, Claudia
TI  - CRISPR/Cas9 deletions induce adverse on-target genomic effects leading to functional DNA in human cells
AID  - 10.1101/2021.07.01.450727
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2021.07.01.450727
4099  - http://biorxiv.org/content/early/2022/04/25/2021.07.01.450727.short
4100  - http://biorxiv.org/content/early/2022/04/25/2021.07.01.450727.full
AB  - The CRISPR/Cas9 system is widely used to permanently delete genomic regions by inducing double-strand breaks via dual guide RNAs. However, on-target consequences of Cas9 deletion events have yet to be fully investigated. To characterize Cas9-induced genotypic abnormalities in human cells, we utilized an innovative droplet-based target enrichment approach followed by long-read sequencing and coupled it to a customized de novo sequence assembly. This approach enabled us to dissect the sequence content at kilobase scale within an on-target genomic locus. We here describe extensive genomic disruptions by Cas9, involving a genomic duplication and inversion of the target region as well as integrations of exogenous DNA and interchromosomal DNA fragment rearrangements at the double-strand break sites often at the same time. Although these events altered the genomic composition of the on-target region, we found that the aberrant DNA fragments are still functional, marked by active histones and bound by RNA polymerase III. In HAP1 cells, the integration of the target-derived fragments accelerated cell proliferation in deletion clones. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations and rationalize extra caution when interpreting results from a deletion event.Competing Interest StatementThe authors have declared no competing interest.ΔttRNA gene locus deletionΔiintergenic region deletionBPbreakpointCas9CRISPR-associated protein 9CRISPRclustered regularly interspaced short palindromic repeatsctrlnon-targeting controlDCdaughter celldMDAdroplet multiple displacement amplificationDSBdouble-strand breakE. coliEscherichia coligRNAguide RNAH3K4me3histone 3 lysine 4 trimethylationHAP1chronic myeloid leukemia cell lineHepG2hepatocellular carcinomaLRSlong-read sequencingMMEJmicrohomology-mediated end joiningNHEJnonhomologous end joiningntnon-transfected controlONTOxford Nanopore TechnologyPAMprotospacer adjacent motifPol IIIRNA polymerase IIItRNAtransfer RNA