CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations

Cullot, Grégoire; Boutin, Julian; Toutain, Jérôme; Prat, Florence; Pennamen, Perrine; Rooryck, Caroline; Teichmann, Martin; Rousseau, Emilie; Lamrissi-Garcia, Isabelle; Guyonnet‐Duperat, Véronique; Bibeyran, Alice; Lalanne, Magalie; Prouzet‐Mauléon, Valérie; Turcq, Béatrice; Ged, Cécile; Blouin, Jean-Marc; Richard, Emmanuel; Dabernat, Sandrine; Moreau‐Gaudry, François; Bedel, Aurélie

doi:10.1038/s41467-019-09006-2

Cited by 342 publications

(285 citation statements)

References 65 publications

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“…However, larger than expected deletions that are suggested by our FISH results ( Fig. S6) and others' reports [32][33][34] , would be missed. Although a single ddXR assay cannot measure all possible excision sizes and rearrangements, further adjustments, similar to the inversion assay, can be easily designed to quantify specific rearrangements of interest once they are defined.…”

Section: Discussionsupporting

confidence: 43%

Rapid, precise quantification of large DNA excisions and inversions by ddPCR

Watry

Feliciano

Gjoni

et al. 2020

Preprint

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The excision of genomic sequences using paired CRISPR-Cas nucleases is a powerful tool to study gene function, create disease models and holds promise for therapeutic gene editing. However, our understanding of the factors that favor efficient excision is limited by the lack of a rapid, accurate measurement of DNA excision outcomes that is free of amplification bias. Here, we introduce ddXR (droplet digital PCR eXcision Reporter), a method that enables the accurate and sensitive detection of excisions and inversions independent of length. The method can be completed in a few hours without the need for next-generation sequencing. The ddXR method uncovered unexpectedly high rates of large (>20 kb) excisions and inversions, while also revealing a surprisingly low dependence on linear distance, up to 170 kb. We further modified the method to measure precise repair of excision junctions and allele-specific excision, with important implications for disease modeling and therapeutic gene editing.

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Section: Discussionsupporting

confidence: 43%

Rapid, precise quantification of large DNA excisions and inversions by ddPCR

Watry

Feliciano

Gjoni

et al. 2020

Preprint

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“…Beyond its impact on biological research, CRISPR-based approaches have been considered for various applications in medicine, from reparative editing of primary cells to the development of new strategies for treating a variety of genetic diseases, including cancer. However, several clinical trials based on CRISPR technology have been deferred due to significant potential risks, including off-target effects 2,3,4 , generation of unexpected chromosomal alterations 5 and potential immunogenicity 6 . Other studies have demonstrated that double stranded breaks (DSBs) induced by CRISPR-Cas9 during gene knockout (KO) can lead to DNA damage response, whose level is associated with the copy number of the targeted gene.…”

mentioning

confidence: 99%

Integrated computational and experimental identification of p53, KRAS and VHL mutant selection associated with CRISPR-Cas9 editing

Sinha

Barbosa

Cheng

et al. 2018

Preprint

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Recent studies have reported that CRISPR-Cas9 gene editing induces a p53-dependent DNA damage response in primary cells, which may select for cells with oncogenic p53 mutations 11,12 .It is unclear whether these CRISPR-induced changes are applicable to different cell types, and whether CRISPR gene editing may select for other oncogenic mutations. Addressing these questions, we analyzed genome-wide CRISPR and RNAi screens to systematically chart the mutation selection potential of CRISPR knockouts across the whole exome. Our analysis suggests that CRISPR gene editing can select for mutants of KRAS and VHL, at a level comparable to that reported for p53. These predictions were further validated in a genome-wide manner by analyzing independent CRISPR screens and patients' tumor data. Finally, we performed a new set of pooled and arrayed CRISPR screens to evaluate the competition between CRISPR-edited isogenic p53 WT and mutant cell lines, which further validated our predictions.In summary, our study systematically charts and points to the potential selection of specific cancer driver mutations during CRISPR-Cas9 gene editing.

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“…Successful G1-variant knock-in and cassette removal were confirmed by Sanger sequencing (Figure 1A, Figure S1A-C). Because CRISPR-mediated genome editing can occasionally induce chromosomal changes 22 , we verified that our genome-edited iPSCs maintained a normal karyotype (Figure S1D-E).…”

Section: Resultsmentioning

confidence: 79%

ProfilingAPOL1Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics

Liu

Radmanesh

Chung

et al. 2019

Preprint

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Running Title: APOL1 Variant Organoid scRNA-seq Word Count: 205 (Abstract), 1736 (Main Text, excluding Methods)ABSTRACT Background DNA variants in APOL1 associate with kidney disease, but the pathophysiological mechanisms remain incompletely understood. Model organisms lack the APOL1 gene, limiting the degree to which disease states can be recapitulated. Here we present single-cell RNA sequencing (scRNAseq) of genome-edited human kidney organoids as a platform for profiling effects of APOL1 risk variants in diverse nephron cell types. MethodsWe performed footprint-free CRISPR-Cas9 genome editing of human induced pluripotent stem cells (iPSCs) to knock in APOL1 high-risk G1 variants at the native genomic locus. iPSCs were differentiated into kidney organoids, treated with vehicle, IFN-g, or the combination of IFN-g and tunicamycin, and analyzed with scRNA-seq to profile cell-specific changes in differential gene expression patterns, compared to isogenic G0 controls. ResultsBoth G0 and G1 iPSCs differentiated into kidney organoids containing nephron-like structures with glomerular epithelial cells, proximal tubules, distal tubules, and endothelial cells.Organoids expressed detectable APOL1 only after exposure to IFN-g. scRNA-seq revealed cell type-specific differences in G1 organoid response to APOL1 induction. Additional stress of tunicamycin exposure led to increased glomerular epithelial cell dedifferentiation in G1 organoids. ConclusionsSingle-cell transcriptomic profiling of human genome-edited kidney organoids expressing APOL1 risk variants provides a novel platform for studying the pathophysiology of APOL1mediated kidney disease.

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CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations

Cited by 342 publications

References 65 publications

Rapid, precise quantification of large DNA excisions and inversions by ddPCR

Rapid, precise quantification of large DNA excisions and inversions by ddPCR

Integrated computational and experimental identification of p53, KRAS and VHL mutant selection associated with CRISPR-Cas9 editing

ProfilingAPOL1Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics

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