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Mol Cell. 2019 Jun 6;74(5):936-950.e5. doi: 10.1016/j.molcel.2019.03.014. Epub 2019 Apr 8.

Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas.

Author information

1
Department of Molecular Biology and Genetics, Cornell University, 253 Biotechnology Building, Ithaca, NY 14853, USA.
2
Department of Biological Chemistry, University of Michigan, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA.
3
Department of Molecular Biology and Genetics, Cornell University, 253 Biotechnology Building, Ithaca, NY 14853, USA; State Key Laboratory of Natural Medicines, Department of Pharmacology, China Pharmaceutical University, Nanjing 210009, China.
4
Murdoch Children's Research Institute, Flemington Rd., Parkville, VIC 3052, Australia; Department of Paediatrics, University of Melbourne, Parkville, VIC 3052, Australia.
5
Department of Biological Chemistry, University of Michigan, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA; Department of Computational Medicine and Bioinformatics, University of Michigan, 100 Washtenaw Avenue, Ann Arbor, MI 48109, USA.
6
Department of Molecular Biology and Genetics, Cornell University, 253 Biotechnology Building, Ithaca, NY 14853, USA. Electronic address: ailong.ke@cornell.edu.
7
Department of Biological Chemistry, University of Michigan, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA. Electronic address: yzhangbc@med.umich.edu.

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.

KEYWORDS:

CRISPR-Cas; Cas3; Cascade; RNA-guided; chromosome; embryonic stem cell; genome editing; large genome deletion; long-range; type I CRISPR

PMID:
30975459
PMCID:
PMC6555677
[Available on 2020-06-06]
DOI:
10.1016/j.molcel.2019.03.014

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