CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations

Höijer, Ida; Emmanouilidou, Anastasia; Östlund, Rebecka; Schendel, Robin van; Bozorgpana, Selma; Tijsterman, Marcel; Feuk, Lars; Gyllensten, Ulf; Hoed, Marcel den; Ameur, Adam

doi:10.1038/s41467-022-28244-5

Cited by 98 publications

(66 citation statements)

References 57 publications

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“…For our PCSK9 sgRNA design, in silico off-target analysis showed no potential off-target sites with less than four mismatched nucleotides and only 21 potential off-target sites with four mismatches, highlighting the high degree of specificity of our design. On the other hand, we did not detect any large structural variations at the edited PCSK9 locus in our work, contrarily to previous observations for direct ribonucleoprotein (RNP) delivery (Höijer et al, 2022). This might be due to the lower amount of RNPs delivered by the engineered EVs, suggesting that EV-based delivery could have a potential advantage compared to other vehicles in circumventing the unwanted effects often observed with CRISPR/Cas9.…”

Section:  Discussionsupporting

confidence: 60%

Engineered Cas9 extracellular vesicles as a novel gene editing tool

Osteikoetxea

Silva

Lázaro-Ibáñez

et al. 2022

J of Extracellular Vesicle

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Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing.

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Section:  Discussionsupporting

confidence: 60%

Engineered Cas9 extracellular vesicles as a novel gene editing tool

Osteikoetxea

Silva

Lázaro-Ibáñez

et al. 2022

J of Extracellular Vesicle

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“…In terms of mutation types (Figure 3C ) and homology at the junctions (Figure 3E ), the two sequence methods generate comparable footprints, with the exception of deletions with insertions (delins), which are more frequently found in PacBio data (Figure 3C ). While the additional mutations detected in PacBio versus Illumina sequencing on these CRISPR sites in mES cells may appear relatively modest, research has shown that such large deletions may occur more frequently in certain cell types and species and that long-read sequencing provides a powerful method to detect undesired genome modifications ( 28 ).…”

Section: Resultsmentioning

confidence: 99%

SIQ: easy quantitative measurement of mutation profiles in sequencing data

Schendel

Schimmel

Tijsterman

2022

NAR Genomics and Bioinformatics

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With the emergence of CRISPR-mediated genome editing, there is an increasing desire for easy-to-use tools that can process and overview the spectra of outcomes. Here, we present Sequence Interrogation and Quantification (SIQ), a simple-to-use software tool that enables researchers to retrieve, data-mine and visualize complex sets of targeted sequencing data. SIQ can analyse Sanger sequences but specifically benefit the processing of short- and long-read next-generation sequencing data (e.g. Illumina and PacBio). SIQ facilitates their interpretation by establishing mutational profiles, with a focus on event classification such as deletions, single-nucleotide variations, (templated) insertions and tandem duplications. SIQ results can be directly analysed and visualized via SIQPlotteR, an interactive web tool that we made freely available. Using insightful tornado plot visualizations as outputs, we illustrate that SIQ readily identifies sequence- and repair pathway-specific mutational signatures in a variety of model systems, such as nematodes, plants and mammalian cell culture.

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“…Recent technological advances in CRISPR/Cas systems are making it possible to induce nucleotide substitutions of interest in a target gene (a technique named ‘allele replacement’) and chromosomal rearrangements [ 104 , 105 ], as reviewed by Ansai and Kitano [ 104 ] in this issue. Although these genome editing technologies often have off-target effects [ 106 ], we should eventually be able to remove such undesirable mutations by backcrossing or future technological advances.…”

Section: How To Go Beyond Candidate Loci To Identify Causative Genes ...mentioning

confidence: 99%

Genetic basis of speciation and adaptation: from loci to causative mutations

Kitano

Ishikawa

Ravinet

et al. 2022

Phil. Trans. R. Soc. B

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Does evolution proceed in small steps or large leaps? How repeatable is evolution? How constrained is the evolutionary process? Answering these long-standing questions in evolutionary biology is indispensable for both understanding how extant biodiversity has evolved and predicting how organisms and ecosystems will respond to changing environments in the future. Understanding the genetic basis of phenotypic diversification and speciation in natural populations is key to properly answering these questions. The leap forward in genome sequencing technologies has made it increasingly easier to not only investigate the genetic architecture but also identify the variant sites underlying adaptation and speciation in natural populations. Furthermore, recent advances in genome editing technologies are making it possible to investigate the functions of each candidate gene in organisms from natural populations. In this article, we discuss how these recent technological advances enable the analysis of causative genes and mutations and how such analysis can help answer long-standing evolutionary biology questions. This article is part of the theme issue ‘Genetic basis of adaptation and speciation: from loci to causative mutations’.

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CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations

Cited by 98 publications

References 57 publications

Engineered Cas9 extracellular vesicles as a novel gene editing tool

Engineered Cas9 extracellular vesicles as a novel gene editing tool

SIQ: easy quantitative measurement of mutation profiles in sequencing data

Genetic basis of speciation and adaptation: from loci to causative mutations

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