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- Geng, KY, et al.
(författare)
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Target-enriched nanopore sequencing and de novo assembly reveals co-occurrences of complex on-target genomic rearrangements induced by CRISPR-Cas9 in human cells
- 2022
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Ingår i: Genome research. - : Cold Spring Harbor Laboratory. - 1549-5469 .- 1088-9051. ; 32:10, s. 1876-1891
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Tidskriftsartikel (refereegranskat)abstract
- The CRISPR-Cas9 system is widely used to permanently delete genomic regions via dual guide RNAs. Genomic rearrangements induced by CRISPR-Cas9 can occur, but continuous technical developments make it possible to characterize complex on-target effects. We combined an innovative droplet-based target enrichment approach with 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 the allelic co-occurrence of a genomic duplication and inversion of the target region, as well as integrations of exogenous DNA and clustered interchromosomal DNA fragment rearrangements. Furthermore, we found that these genomic alterations led to functional aberrant DNA fragments and can alter cell proliferation. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations, and introduce a data-driven workflow enabling detailed dissection of the on-target sequence content with superior resolution.
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- Sych, T, et al.
(författare)
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How Does Liquid-Liquid Phase Separation in Model Membranes Reflect Cell Membrane Heterogeneity?
- 2021
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Ingår i: Membranes. - : MDPI AG. - 2077-0375. ; 11:5
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Tidskriftsartikel (refereegranskat)abstract
- Although liquid–liquid phase separation of cytoplasmic or nuclear components in cells has been a major focus in cell biology, it is only recently that the principle of phase separation has been a long-standing concept and extensively studied in biomembranes. Membrane phase separation has been reconstituted in simplified model systems, and its detailed physicochemical principles, including essential phase diagrams, have been extensively explored. These model membrane systems have proven very useful to study the heterogeneity in cellular membranes, however, concerns have been raised about how reliably they can represent native membranes. In this review, we will discuss how phase-separated membrane systems can mimic cellular membranes and where they fail to reflect the native cell membrane heterogeneity. We also include a few humble suggestions on which phase-separated systems should be used for certain applications, and which interpretations should be avoided to prevent unreliable conclusions.
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