Item Type: | Preprint |
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Title: | A homology independent sequence replacement strategy in human cells using a CRISPR nuclease |
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Creators Name: | Danner, E.W. and Lebedin, M. and de la Rosa, K. and Kühn, R. |
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Abstract: | Precision genomic alterations largely rely on Homology Directed Repair (HDR), but targeting without homology using the Non-Homologous End Joining (NHEJ) pathway has gained attention as a promising alternative. Previous studies demonstrated precise insertions formed by the ligation of donor DNA into a targeted genomic double strand break in both dividing and non-dividing cells. Here we extend this idea and use NHEJ repair to replace genomic segments with donor sequences; we name this method 'Replace' editing (Rational end-joining protocol deliveringa targeted sequence exchange). Using CRISPR/Cas9 we create two genomic breaks and ligate a donor sequence in-between. This exchange of a genomic for a donor sequence uses neither microhomology nor homology arms. We target four loci and show successful exchange of exons in 16% to 54% of cells. Using linear amplification methods and deep sequencing pipelines we quantify the diversity of outcomes following Replace editing and profile mutations formed at the ligated interfaces. The ability to replace exons or other genomic sequences in cells not efficiently modified by HDR holds promise for both basic research and medicine. |
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Source: | bioRxiv |
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Title of Book: | A Homology Independent Sequence Replacement Strategy in Human Cells Using a CRISPR Nuclease |
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Publisher: | Cold Spring Harbor Laboratory Press |
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Article Number: | 2020.05.11.088252 |
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Date: | 12 May 2020 |
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Official Publication: | https://doi.org/10.1101/2020.05.11.088252 |
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Related to: | |
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