Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas.
Mol Cell
; 74(5): 936-950.e5, 2019 06 06.
Article
em En
| MEDLINE
| ID: mdl-30975459
ABSTRACT
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.
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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Deleção de Sequência
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RNA Guia de Cinetoplastídeos
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Sistemas CRISPR-Cas
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Células-Tronco Embrionárias Humanas
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article