A homology independent sequence replacement strategy in human cells using a CRISPR nuclease
| Autor: | Ralf Kühn, Kathrin de la Rosa, Eric Danner, Mikhail Lebedin |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Cancer Research
DNA End-Joining Repair Genotype Computational biology Homology (biology) Deep sequencing Homology directed repair Exon chemistry.chemical_compound Genes Reporter Cell Line Tumor replace editing CRISPR Humans exon replacement lcsh:QH301-705.5 DNA Polymerase beta Gene Editing Nuclease biology Cas9 Research High-Throughput Nucleotide Sequencing Exons chemistry lcsh:Biology (General) Genetic Loci biology.protein CRISPR-Cas Systems Technology Platforms Nucleic Acid Amplification Techniques DNA RNA Guide Kinetoplastida Research Article |
| Zdroj: | Open Biology Open Biology, Vol 11, Iss 1 (2021) |
| Popis: | 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 (Rationalend-joiningprotocol deliveringatargeted sequenceexchange). 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. |
| Databáze: | OpenAIRE |
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