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J Biol Chem. 2014 Aug 1;289(31):21312-24. doi: 10.1074/jbc.M114.564625. Epub 2014 Jun 6.

Characterization of genomic deletion efficiency mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells.

Author information

1
From the Harvard Medical School.
2
From the Harvard Medical School, the Division of Hematology/Oncology, Boston Children's Hospital, the Department of Pediatric Oncology, Dana-Farber Cancer Institute.
3
the Division of Hematology/Oncology, Boston Children's Hospital.
4
From the Harvard Medical School, the Division of Hematology, Brigham and Women's Hospital, and.
5
the Division of Hematology, Brigham and Women's Hospital, and.
6
From the Harvard Medical School, the Division of Hematology/Oncology, Boston Children's Hospital, the Department of Pediatric Oncology, Dana-Farber Cancer Institute, the Division of Hematology, Brigham and Women's Hospital, and.
7
From the Harvard Medical School, the Division of Hematology/Oncology, Boston Children's Hospital, the Department of Pediatric Oncology, Dana-Farber Cancer Institute, the Howard Hughes Medical Institute, Boston, Massachusetts 02115 Stuart_Orkin@dfci.harvard.edu.

Abstract

The clustered regularly interspaced short [corrected] palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements.

KEYWORDS:

CRISPR; Cas9; Gene Expression; Gene Knock-out; Gene Regulation; Genetics; Genome Engineering; Genomic Deletion; Genomics

PMID:
24907273
PMCID:
PMC4118095
DOI:
10.1074/jbc.M114.564625
[Indexed for MEDLINE]
Free PMC Article

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