Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas
| Autor: | Ailong Ke, Yibei Xiao, Jaewon Heo, Max J. Gramelspacher, Peter L. Freddolino, Yan Zhang, Sara E. Howden, Zhonggang Hou, Adam E. Dolan |
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| Rok vydání: | 2018 |
| Předmět: |
Base pair
Human Embryonic Stem Cells Computational biology Biology Genome 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Genome editing Escherichia coli HAP1 cells CRISPR Humans Molecular Biology 030304 developmental biology Ribonucleoprotein Sequence Deletion Gene Editing 0303 health sciences Genome Human RNA Cell Biology Genomics Endonucleases chemistry Ribonucleoproteins CRISPR-Cas Systems 030217 neurology & neurosurgery DNA RNA Guide Kinetoplastida |
| Zdroj: | Molecular cell. 74(5) |
| ISSN: | 1097-4164 |
| Popis: | Summary CRISPR-Cas systems enable microbial adaptive immunity and provide eukaryotic genome editing tools. These tools employ a single effector enzyme of type II or V CRISPR to generate RNA-guided, precise genome breaks. Here we demonstrate the feasibility of using type I CRISPR-Cas to effectively introduce a spectrum of long-range chromosomal deletions with a single RNA guide in human embryonic stem cells and HAP1 cells. Type I CRISPR systems rely on the multi-subunit ribonucleoprotein (RNP) complex Cascade to identify DNA targets and on the helicase-nuclease enzyme Cas3 to degrade DNA processively. With RNP delivery of T. fusca Cascade and Cas3, we obtained 13%–60% editing efficiency. Long-range PCR-based and high-throughput-sequencing-based lesion analyses reveal that a variety of deletions, ranging from a few hundred base pairs to 100 kilobases, are created upstream of the target site. These results highlight the potential utility of type I CRISPR-Cas for long-range genome manipulations and deletion screens in eukaryotes. |
| Databáze: | OpenAIRE |
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