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On- and off-target effects of paired CRISPR-Cas nickase in primary human cells - Illumina

ABSTRACT: On- and off-target effects of paired CRISPR-Cas nickase in primary human cells - Illumina

PROVIDER: PRJNA1009854 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR25765254_1.fastq.gz	Fastqsanger.gz
	SRR25765254_2.fastq.gz	Fastqsanger.gz
	SRR25765255_1.fastq.gz	Fastqsanger.gz
	SRR25765255_2.fastq.gz	Fastqsanger.gz
	SRR25765256_1.fastq.gz	Fastqsanger.gz

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On- and off-target effects of paired CRISPR-Cas nickase in primary human cells - Illumina

Project description: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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On- and off-target effects of paired CRISPR-Cas nickase in primary human cells [Amplicon]

Project description:Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in 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-strand 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 aberrations 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 paired nickase editing. While 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, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects, but still leave substantial chromosomal aberrations at on-target sites.

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On- and off-target effects of paired CRISPR-Cas nickase in primary human cells

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Project description:On- and off-target effects of paired CRISPR-Cas nickase in primary human cells - long read