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| Status |
Public on Mar 15, 2024 |
| Title |
On- and off-target effects of paired CRISPR-Cas nickase in primary human cells - long read |
| Organism |
Homo sapiens |
| Experiment type |
Other
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| Summary |
Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in therapeutic genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double nickase-based strategies to generate staggered DNA double breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-Seq pipeline, D-CAST, to characterize chromosomal rearrangements induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from epidermolysis bullosa patients. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following single or paired Cas9-based nickase editing. Conversely, whereas single nickase applications did not result in gross genomic aberrations, the double nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects, but still leave substantial chromosomal rearrangements at on-target sites.
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| Overall design |
Primary patient-derived keratinocytes were treated with Cas9 nucleases and single- and double-nickases. On-target effects were assessed using long-read (LR) sequencing.
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| Contributor(s) |
Andrieux G, Klermund J, Boerries M, Cathomen T |
| Citation missing |
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| |
| Submission date |
Feb 02, 2024 |
| Last update date |
Mar 15, 2024 |
| Contact name |
Geoffroy Andrieux |
| Organization name |
University clinics Freiburg
|
| Street address |
Breisacherstr 153
|
| City |
Freiburg |
| ZIP/Postal code |
79110 |
| Country |
Germany |
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| Platforms (1) |
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| Samples (14)
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| GSM8060256 |
Long-read sequencing of COL7A1 on-target site on sample treated with Cas9 nuclease + ssODN g1 |
| GSM8060257 |
Long-read sequencing of COL7A1 on-target site on sample treated with Cas9 nuclease + ssODN g2 |
| GSM8060258 |
Long-read sequencing of COL7A1 on-target site on sample treated with Cas9 double nickase + ssODN |
| GSM8060259 |
Long-read sequencing of COL7A1 on-target site of untreated control + ssODN |
| GSM8060260 |
Long-read sequencing of COL17A1 on-target site on sample treated with Cas9 nuclease g3 |
| GSM8060261 |
Long-read sequencing of COL17A1 on-target site on sample treated with Cas9 nuclease g4 |
| GSM8060262 |
Long-read sequencing of COL17A1 on-target site on sample treated with Cas9 double nickase |
| GSM8060263 |
Long-read sequencing of COL17A1 on-target site of untreated control |
| GSM8060264 |
Long-read sequencing of COL17A1 on-target site on sample treated with Cas9 double nickase + ssODN |
| GSM8060265 |
Long-read sequencing of COL17A1 on-target site of untreated control + ssODN |
| GSM8060266 |
Long-read sequencing of LAMA3 on-target site on sample treated with Cas9 nuclease g5 |
| GSM8060267 |
Long-read sequencing of LAMA3 on-target site on sample treated with Cas9 nuclease g6 |
| GSM8060268 |
Long-read sequencing of LAMA3 on-target site on sample treated with Cas9 double nickase |
| GSM8060269 |
Long-read sequencing of LAMA3 on-target site of untreated control |
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| This SubSeries is part of SuperSeries: |
| GSE241780 |
On- and off-target effects of paired CRISPR-Cas nickase in primary human cells |
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| Relations |
| BioProject |
PRJNA1072631 |
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